Inflammation and cancer : the role of extracellular enolase-1

Extracellular matrix degradation is one of the crucial steps in cancer cell invasion and spreading. A number of proteases, including plasmin, mediate disruption of stromal barriers and basement membrane and thus facilitate tumor cell movement. Formation of plasmin is a result of the plasminogen (PLG) activation cascade, which involves PLG activators and receptors. Enolase-1 (ENO-1) is one of the plasminogen receptors (PLG-R). It belongs to the so called “moonlighting protein group”, which exhibits various functions at distinct cellular and extracellular sites of the cell. This primary glycolytic enzyme was found to be overexpressed in more than 20 types of human cancer and accounts for enhanced cancer progression and poor clinical outcome. Although numerous studies provide evidence for pro-tumorigenic properties of cytoplasmic ENO-1, the contribution of cell surface bound ENO-1 to cancer progression has not yet been described. Here, we demonstrate increased expression of ENO-1 in different types of human cancer, in particular, in breast ductal carcinoma. Cell fractionation of the breast cancer cells (MDA-MB-231) revealed elevated ENO-1 cell surface levels, which correlated with enhanced migratory and invasive properties of these cells. Overexpression of wild-type ENO-1 increased invasion of MDA-MB-231 cells. This effect was not observed when ENO-1 mutant bearing the mutation in a PLG binding site was overexpressed. Exposure of MDA-MB-231 cells to LPS further potentiated ENO-1 cell surface expression and simultaneously increased release of ENO-1 to the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was diminished upon pretreatment of MDA-MB-231 cells with the Ca2+ chelator BAPTA or an inhibitor of endoplasmic reticulum Ca2+-ATPase pump, cyclopiazonic acid. In line with this observation, STIM1 and ORAI1 were found to regulate LPS-induced ENO-1 cell surface expression and release. Accordingly, pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of breast cancer cells. Collectively, these data reveal the functional consequence of extracellulary localized ENO-1 in cancer cell behaviour and the mechanism which drives ENO-1 exteriorization. Thus, targeting cell surface bound ENO-1 may offer a novel therapeutic strategy in patients suffering from cancer.Der Abbau der extrazellulären Matrix ist einer der entscheidenden Schritte bei der Krebszellinvasion und –ausbreitung. Mehrere Proteasen einschließlich Plasmin vermitteln die Auflösung der stromalen Barrieren und der Basalmembran und erleichtern so die Bewegung der Tumorzellen. Die Plasminbildung ist das Ergebnis der Plasminogen (PLG)- Aktivierungskaskade, die PLG-Aktivatoren und Rezeptoren umfasst. Enolase-1 (ENO-1) ist einer dieser Plasminogenrezeptoren (PLG-R). Sie gehört zur Gruppe der sogenannten „moonlighting“-Proteine, die mehrere Funktionen in unterschiedlichen zellulären und extrazellulären Bereichen der Zelle aufweisen. Dieses primär glykolytische Enzym, das in mehr als 20 humanen Krebsarten überexprimiert ist, ist verantwortlich für ein schnelleres Fortschreiten der Krebserkrankungen und für eine schlechte klinische Prognose. Obwohl zahlreiche Studien die tumorerzeugenden Eigenschaften von zytoplasmatischer ENO-1 beweisen, wurde der Einfluss der oberflächengebundenen ENO-1 noch nicht beschrieben.In dieser Arbeit zeigen wir nun die erhöhte Expression von ENO-1 in verschiedenen humanen Tumortypen, insbesondere im duktalen Brustkarzinom. Die Zellfraktionierung der Brustkrebszellen MDA-MB-231, ergab ein erhöhtes Niveau der ENO-1 an der Zelloberfläche, welches mit den verbesserten Invasions- und Migrationseigenschaften dieser Zellen korreliert. Die Überexpression der Wildtyp- ENO-1 erhöht die Einwanderung der MDA-MB-231. Dieser Effekt konnte nicht beobachtet werden, wenn eine ENO-1-Mutante überexprimiert wurde, die eine Mutation in der PLG-Bindungsstelle aufwies. Wurden die MDA-MB-231 mit LPS behandelt, so wurde die Expression der ENO-1 an der Zelloberfläche weiter verstärkt, was gleichzeitig zu einer erhöhten Freisetzung der ENO-1 in den extrazellulären Raum in Form von Exosomen führte. Diese Effekte waren unabhängig von der de novo Proteinsynthese und benötigten nicht den klassischen Pfad über das endoplasmatische Retikulum und den Golgi-Apparat. Die LPS-gesteuerte Exteriorisation wurde durch eine Vorbehandlung der MDA-MB-231 mit BAPTA einem Ca2+-Chelator oder Cyclopiazonsäure dem Inhibitor der Ca2+-ATPase-Pumpe des endoplasmatischen Retikulums verringert. Dies entsprach der Beobachtung, dass STIM1 und ORAI1 die LPS-induzierte Expression von ENO-1 an der Zelloberfläche und die Freisetzung der ENO-1 regulieren. Dementsprechend reduzierte eine pharmakologische Blockierung oder ein Knockdown von STIM1 oder ORAI1 die faktorabhängige Migration der Brustkrebszellen. Zusammengefasst zeigen diese Daten den Mechanismus auf, der die Exteriorization von ENO-1 steuert und die funktionellen Auswirkungen dieser extrazellulär lokalisierten ENO-1 auf das Verhalten der Krebszellen. Folglich stellt die zelloberflächengebundene ENO-1 das Ziel einer neuen therapeutischen Strategie bei der Behandlung von Krebspatienten dar

When Place Matters: Shuttling of Enolase-1 Across Cellular Compartments

Enolase is a glycolytic enzyme, which catalyzes the inter-conversion of 2-phosphoglycerate to phosphoenolpyruvate. Altered expression of this enzyme is frequently observed in cancer and accounts for the Warburg effect, an adaptive response of tumor cells to hypoxia. In addition to its catalytic function, ENO-1 exhibits other activities, which strongly depend on its cellular and extracellular localization. For example, the association of ENO-1 with mitochondria membrane was found to be important for the stability of the mitochondrial membrane, and ENO-1 sequestration on the cell surface was crucial for plasmin-mediated pericellular proteolysis. The latter activity of ENO-1 enables many pathogens but also immune and cancer cells to invade the tissue, leading further to infection, inflammation or metastasis formation. The ability of ENO-1 to conduct so many diverse processes is reflected by its contribution to a high number of pathologies, including type 2 diabetes, cardiovascular hypertrophy, fungal and bacterial infections, cancer, systemic lupus erythematosus, hepatic fibrosis, Alzheimer’s disease, rheumatoid arthritis, and systemic sclerosis. These unexpected non-catalytic functions of ENO-1 and their contributions to diseases are the subjects of this review

Succinic semialdehyde dehydrogenase deficiency: in vitro and in silico characterization of a novel pathogenic missense variant and analysis of the mutational spectrum of ALDH5A1

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter \u3b3-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including \u3b3-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots

Targeting TMEM16A to reverse vasoconstriction and remodelling in idiopathic PAH

Our systematic analysis of anion channels and transporters in idiopathic pulmonary arterial hypertension (IPAH) showed marked upregulation of the Cl- channel TMEM16A gene.We hypothesised that TMEM16A overexpression might represent a novel vicious circle in the molecular pathways causing PAH.We investigated healthy donor lungs (n=40) and recipient lungs with IPAH (n=38) for the expression of anion channel and transporter genes in small pulmonary arteries and pulmonary arterial smooth muscle cells (PASMC). In IPAH, TMEM16A was strongly upregulated and patch-clamp recordings confirmed an increased Cl- current in PASMC (n=9-10). These cells were depolarised and could be repolarized by TMEM16A inhibitors or knock-down experiments (n=6-10). Inhibition/knock-down of TMEM16A reduced proliferation of IPAH-PASMC (n=6). Conversely, overexpression of TMEM16A in healthy donor PASMC produced an IPAH-like phenotype. Chronic application of benzbromarone in two independent animal models significantly decreased right ventricular pressure and reversed remodelling of established PH.Our findings suggest that increased TMEM16A expression and activity comprise an important pathologic mechanism underlying vasoconstriction and remodelling of pulmonary arteries in PAH. Inhibition of TMEM16A represents a novel therapeutic approach to achieve reverse remodelling in PAH

From Plasminogen to Plasmin: Role of Plasminogen Receptors in Human Cancer

Cell surface-associated proteolysis mediated by plasmin (PLA) is an essential feature of wound healing, angiogenesis and cell invasion, processes that are dysregulated in cancer development, progression and systemic spread. The generation of PLA, initiated by the binding of its precursor plasminogen (PLG) to the cell surface, is regulated by an array of activators, inhibitors and receptors. In this review, we will highlight the importance of the best-characterized components of the PLG/PLA cascade in the pathogenesis of cancer focusing on the role of the cell surface-PLG receptors (PLG-R). PLG-R overexpression has been associated with poor prognosis of cancer patients and resistance to chemotherapy. We will also discuss recent findings on the molecular mechanisms regulating cell surface expression and distribution of PLG-R

When place matters: shuttling of enolase-1 across cellular compartments

Enolase is a glycolytic enzyme, which catalyzes the inter-conversion of 2-phosphoglycerate to phosphoenolpyruvate. Altered expression of this enzyme is frequently observed in cancer and accounts for the Warburg effect, an adaptive response of tumor cells to hypoxia. In addition to its catalytic function, ENO-1 exhibits other activities, which strongly depend on its cellular and extracellular localization. For example, the association of ENO-1 with mitochondria membrane was found to be important for the stability of the mitochondrial membrane, and ENO-1 sequestration on the cell surface was crucial for plasmin-mediated pericellular proteolysis. The latter activity of ENO-1 enables many pathogens but also immune and cancer cells to invade the tissue, leading further to infection, inflammation or metastasis formation. The ability of ENO-1 to conduct so many diverse processes is reflected by its contribution to a high number of pathologies, including type 2 diabetes, cardiovascular hypertrophy, fungal and bacterial infections, cancer, systemic lupus erythematosus, hepatic fibrosis, Alzheimer’s disease, rheumatoid arthritis, and systemic sclerosis. These unexpected non-catalytic functions of ENO-1 and their contributions to diseases are the subjects of this review

Targeting GLI Transcription Factors in Cancer

Aberrant activation of hedgehog (Hh) signaling has been observed in a wide variety of tumors and accounts for more than 25% of human cancer deaths. Inhibitors targeting the Hh signal transducer Smoothened (SMO) are widely used and display a good initial efficacy in patients suffering from basal cell carcinoma (BCC); however, a large number of patients relapse. Though SMO mutations may explain acquired therapy resistance, a growing body of evidence suggests that the non-canonical, SMO-independent activation of the Hh pathway in BCC patients can also account for this adverse effect. In this review, we highlight the importance of glioma-associated oncogene (GLI) transcription factors (the main downstream effectors of the canonical and the non-canonical Hh cascade) and their putative role in the regulation of multiple oncogenic signaling pathways. Moreover, we discuss the contribution of the Hh signaling to malignant transformation and propose GLIs as central hubs in tumor signaling networks and thus attractive molecular targets in anti-cancer therapies

Protein arginine methyltransferase 5 mediates enolase-1 cell surface trafficking in human lung adenocarcinoma cells

Objectives: Enolase-l-dependent cell surface proteolysis plays an important role in cell invasion. Although enolase-1 (Eno-1), a glycolytic enzyme, has been found on the surface of various cells, the mechanism responsible for its exteriorization remains elusive. Here, we investigated the involvement of post-translational modifications (PTMs) of Eno-1 in its lipopolysaccharide (LPS)-triggered trafficking to the cell surface. Results: We found that stimulation of human lung adenocarcinoma cells with LPS triggered the monomethylation of arginine 50 (R5Ome) within Eno-1. The Eno-1R5Ome was confirmed by its interaction with the tudor domain (TD) from TD-containing 3 (TDRD3) protein recognizing methylarginines. Substitution of R50 with lysine (R50K) reduced Eno-1 association with epithelial caveolar domains, thereby diminishing its exteriorization. Similar effects were observed when pharmacological inhibitors of arginine methyltransferases were applied. Protein arginine methyltransferase 5 (PRMT5) was identified to be responsible for Eno-1 methylation. Overexpression of PRMT5 and caveolin-1 enhanced levels of membrane-bound extracellular Eno-1 and, conversely, pharmacological inhibition of PRMT5 attenuated Eno-1 cell-surface localization. Importantly, Eno1R5Ome was essential for cancer cell motility since the replacement of Eno-1 R50 by lysine or the suppression of PRMT 5 activity diminished Eno-l-triggered cell invasion. Conclusions: LPS-triggered Eno-1R5Ome enhances Eno-1 cell surface levels and thus potentiates the invasive properties of cancer cells. Strategies to target Eno-1R5Ome may offer novel therapeutic approaches to attenuate tumor metastasis in cancer patients