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Gary Thomas - One of the best experts on this subject based on the ideXlab platform.
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inhibition of Furin mediated processing results in suppression of astrocytoma cell growth and invasiveness
Clinical Cancer Research, 2019Co-Authors: Javier Mercapide, Daniele Bassi, Gary Thomas, Ricardo Lopez De Cicco, Javier S Castresana, Andres J P KleinszantoAbstract:Purpose: Astrocytoma arises in the central nervous system as a tumorof great lethality, in part because of the invasive potential of the neoplastic cells that are able to release extracellular matrix-degrading enzymes. Furin convertase activates several precursor matrix metalloproteases involved in the breakdown of the extracellular matrix. In the present study inhibition of Furin was achieved by gene transfer of α 1 -antitrypsin Portland (PDX) cDNA. Experimental Design: This Furin inhibitor was transfected into two tumorigenic astrocytoma cell lines. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, and proliferation assays, as well as by investigating impairment of Furin substrate processing. Results: Expression of PDX prevented the s.c. growth of the transfected cells. Invasion assays demonstrated that PDX-transfected cells exhibited a reduced invasive ability in vitro and in vivo . Furthermore, s.c. growth of PDX transfectant xenotransplants showed a significant reduction in size that coincided with a significant decrease of the in vitro doubling time and of the in vivo cell proliferation ability. Additional studies showed that the Furin substrates insulin-like growth factor IR, transforming growth factor β and membrane type 1-matrix metalloprotease were not activated in PDX-expressing astrocytoma cells. Conclusions: PDX expression in astrocytoma cells demonstrated a direct mechanistic link between Furin inhibition, and decreased astrocytoma proliferation and invasive ability. Because Furin inhibition inhibits both invasiveness and cell growth in astrocytoma, Furin should be considered a promising target for glioblastoma therapy.
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the mechanism by which a propeptide encoded ph sensor regulates spatiotemporal activation of Furin
Journal of Biological Chemistry, 2013Co-Authors: Danielle M Williamson, Gary Thomas, Johannes Elferich, Parvathy Ramakrishnan, Ujwal ShindeAbstract:The proprotein convertase Furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the Furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates Furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of Furin. Structural analyses and binding experiments comparing the wild-type Furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks Furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for Furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of Furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific Furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.
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synthetic small molecule Furin inhibitors derived from 2 5 dideoxystreptamine
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Guansheng Jiao, Lynne Cregar, Jinzhi Wang, Sherri Z Millis, Cho Tang, Sean Omalley, Alan T Johnson, Sina Sareth, Jason Larson, Gary ThomasAbstract:Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of Furin may provide a feasible and promising approach for therapeutic intervention of Furin-mediated disease mechanisms. Here, we report on a class of small molecule Furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of Furin inhibitors with nanomolar range potency against Furin when assayed in a biochemical cleavage assay. Moreover, a subset of these Furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by Furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of Furin and to be relatively specific for Furin. Molecular modeling revealed that these inhibitors may target the active site of Furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-Lys-Arg-CH2Cl. The compounds presented here are bona fide synthetic small molecule Furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying Furin action and potential therapeutics agents for Furin-dependent diseases.
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proprotein convertase Furin interacts with and cleaves pro adamts4 aggrecanase 1 in the trans golgi network
Journal of Biological Chemistry, 2004Co-Authors: Ping Wang, Gary Thomas, Micky D Tortorella, Kristen England, A M Malfait, Elizabeth C ArnerAbstract:Abstract A member of the A disintegrin and metalloproteinase domain with thrombospondin type-1 motifs (ADAMTS-4) protease family can efficiently cleave aggrecan at several sites detected in joints of osteoarthritic patients. Although recent studies have shown that removal of the prodomain of ADAMTS4 is critical for its ability to degrade aggrecan, the cellular mechanisms for its processing and trafficking remain unclear. In this study, by using both Furin-specific inhibitor and RNA interference technique, we demonstrate that Furin plays an important role in the intracellular removal of ADAMTS4 prodomain. Further, we demonstrate that proADAMTS4 can be processed by means of multiple Furin recognition sites: 206RPRR209, 209RAKR212, or 211KR212. The processing of proADAMTS4 was completely blocked by brefeldin A treatment, suggesting that processing occurs in the trans-Golgi network. Indeed, ADAMTS4 is co-localized with Furin in trans-Golgi network. Interestingly, the pro form of ADAMTS4, not its mature one, co-precipitates with Furin, suggesting that Furin physically interacts with the prodomain of ADAMTS-4. In addition, our evidence suggests that a Furin-independent pathway may also contribute to the activation of ADAMTS4. These results indicate that the activation mechanism for ADAMTS4 can be targeted for therapeutical intervention against this enzyme.
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the ordered and compartment specific autoproteolytic removal of the Furin intramolecular chaperone is required for enzyme activation
Journal of Biological Chemistry, 2002Co-Authors: Eric D Anderson, Sean S Molloy, Francois Jean, Satoko Shimamura, Gary ThomasAbstract:Abstract The propeptide of Furin has multiple roles in guiding the activation of the endoprotease in vivo. The 83-residue N-terminal propeptide is autoproteolytically excised in the endoplasmic reticulum (ER) at the consensus Furin site, -Arg104-Thr-Lys-Arg107↓-, but remains bound to Furin as a potent autoinhibitor. Furin lacking the propeptide is ER-retained and proteolytically inactive. Co-expression with the propeptide, however, restores trans-Golgi network (TGN) localization and enzyme activity, indicating that the Furin propeptide is an intramolecular chaperone. Blocking this step results in localization to the ER-Golgi intermediate compartment (ERGIC)/cis-Golgi network (CGN), suggesting the ER and ERGIC/CGN recognize distinct Furin folding intermediates. Following transport to the acidified TGN/endosomal compartments, Furin cleaves the bound propeptide at a second, internal P1/P6 Arg site (-Arg-Gly-Val72-Thr-Lys-Arg75↓-) resulting in propeptide dissociation and enzyme activation. Cleavage at Arg75, however, is not required for proper Furin trafficking. Kinetic analyses of peptide substrates indicate that the sequential pH-modulated propeptide cleavages result from the differential recognition of these sites by Furin. Altering this preference by converting the internal site to a canonical P1/P4 Arg motif (Val72 → Arg) caused ER retention and blocked activation of Furin, demonstrating that the structure of the Furin propeptide mediates folding of the enzyme and directs its pH-regulated, compartment-specific activation in vivo.
Wolfgang Garten - One of the best experts on this subject based on the ideXlab platform.
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potent inhibitors of Furin and Furin like proprotein convertases containing decarboxylated p1 arginine mimetics
Journal of Medicinal Chemistry, 2010Co-Authors: Gero L Becker, Wolfgang Garten, Iris Lindberg, Manuel E Than, Yinghui Lu, Frank Sielaff, Sophie Routhier, Torsten SteinmetzerAbstract:Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related Furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analogue peptidic inhibitors, we could identify highly potent Furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits Furin with a Ki value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Molecular modeling revealed seve...
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two independent targeting signals in the cytoplasmic domain determine trans golgi network localization and endosomal trafficking of the proprotein convertase Furin
The EMBO Journal, 1995Co-Authors: Wolfram Schafer, Hansdieter Klenk, Annemarie Stroh, Susanne Berghofer, J Seiler, M L Kruse, H F Kern, Wolfgang GartenAbstract:Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When Furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of Furin were investigated by mutational analysis of the cytoplasmic tail of Furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of Furin. The membrane-spanning domain of Furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on Furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type Furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-Furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains Furin in the TGN, whereas the YKGL motif acts as a retrieval signal for Furin that has escaped to the cell surface.
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inhibition of Furin mediated cleavage activation of hiv 1 glycoprotein gp160
Nature, 1992Co-Authors: Sabine Hallenberger, Valerie Bosch, Herbert Angliker, Elliott Shaw, Hansdieter Klenk, Wolfgang GartenAbstract:THE envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection1 by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues2. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is Furin3. Furin, a subtilisin-like eukaryotic endoprotease4–6, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site7. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by Furin. Furthermore, we present evidence that peptidyl-chloromethyl ketones that have the Arg-X-Lys/Arg-Arg motif and which are specific inhibitors of Furin3 interfere with cleavage of the HIV glycoprotein and hence its activation and the formation of infectious virus particles.
Gary R Whittaker - One of the best experts on this subject based on the ideXlab platform.
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host cell entry of middle east respiratory syndrome coronavirus after two step Furin mediated activation of the spike protein
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Jean K Millet, Gary R WhittakerAbstract:Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified betacoronavirus causing high morbidity and mortality in humans. The coronavirus spike (S) protein is the main determinant of viral entry, and although it was previously shown that MERS-CoV S can be activated by various proteases, the details of the mechanisms of proteolytic activation of fusion are still incompletely characterized. Here, we have uncovered distinctive characteristics of MERS-CoV S. We identify, by bioinformatics and peptide cleavage assays, two cleavage sites for Furin, a ubiquitously expressed protease, which are located at the S1/S2 interface and at the S2′ position of the S protein. We show that although the S1/S2 site is proteolytically processed by Furin during protein biosynthesis, the S2′ site is cleaved upon viral entry. MERS-CoV pseudovirion infection was shown to be enhanced by elevated levels of Furin expression, and entry could be decreased by Furin siRNA silencing. Enhanced Furin activity appeared to partially override the low pH-dependent nature of MERS-CoV entry. Inhibition of Furin activity was shown to decrease MERS-CoV S-mediated entry, as well as infection by the virus. Overall, we show that MERS-CoV has evolved an unusual two-step Furin activation for fusion, suggestive of a role during the process of emergence into the human population. The ability of MERS-CoV to use Furin in this manner, along with other proteases, may explain the polytropic nature of the virus.
Nabil G. Seidah - One of the best experts on this subject based on the ideXlab platform.
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HIV-induced neuroinflammation: impact of PAR1 and PAR2 processing by Furin.
Cell Death & Differentiation, 2019Co-Authors: Vatsal Sachan, Josee Hamelin, Robert Lodge, Koichiro Mihara, Christopher Power, Benjamin B. Gelman, Morley D. Hollenberg, Éric A. Cohen, Nabil G. SeidahAbstract:HIV-associated neurocognitive disorders (HAND) is a syndrome defined by neurocognitive deficits that are driven by viral neurotoxins, cytokines, free radicals, and proteases expressed in the brain. This neurological disease has also been linked to activation of Protease-Activated Receptors 1 and 2 (PAR1,2). These receptors are highly expressed in the central nervous system and are upregulated in HAND. Secretory basic-amino-acid-specific Proprotein Convertases (PCs), which cleave precursor proteins at basic residues, are also induced in HAND. They are vital for many biological processes including HIV-1 entry into cells. The cytoprotective role of Furin, PC5, and PACE4 has been linked to the presence of a potential PC-cleavage site R41XXXXR46↓ in PAR1. Furthermore, Furin binds PAR1 and both are trapped in the trans-Golgi-network (TGN) as inactive proteins, likely due to the intermediary trafficking role of phospho-Furin acidic cluster sorting protein 1 (PACS1). Nothing is known about PAR2 and its possible recognition by PCs at its putative R31XXXXR36↓ processing site. The present study implicates PACS1 in the retrograde trafficking of PAR1 to the TGN and demonstrates that the cytosolic extreme C-terminal tail of PAR1 contains an acidic phosphorylatable PACS1-sensitive domain. We further show the requirement of Asn47 in PAR1 for its Furin-dependent TGN localization. Our data revealed that Furin is the only convertase that efficiently cleaves PAR2 at Arg36↓. N-glycosylation of PAR2 at Asn30 reduces the efficacy, but enhances selectivity of the Furin cleavage. Finally, in co-cultures comprised of human neuroblastoma SK-N-SH cells (stably expressing PAR1/2 and/or Furin) and HIV-1-infected primary macrophages, we demonstrate that the expression of Furin enhances neuronal cell viability in the context of PAR1- or PAR2-induced neuronal cytotoxicity. The present study provides insights into early stages of HIV-1 induced neuronal injury and the protective role of Furin in neurons co-expressing PAR1 and/or PAR2, as observed in HAND.
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Furin is the primary in vivo convertase of angiopoietin like 3 and endothelial lipase in hepatocytes
Journal of Biological Chemistry, 2013Co-Authors: Rachid Essalmani, John W.m. Creemers, Annik Prat, Delia Susanresiga, Ann Chamberland, Marieclaude Asselin, Maryssa Canuel, Daniel Constam, Dany Gauthier, Nabil G. SeidahAbstract:The proprotein convertases (PCs) Furin, PC5/6, and PACE4 exhibit unique and/or complementary functions. Their knock-out (KO) in mice resulted in strong and specific phenotypes demonstrating that, in vivo, these PCs are unique and essential during development. However, they also exhibit redundant functions. Liver angiopoietin-like 3 (ANGPTL3) inhibits lipolysis by binding to lipoprotein lipases. It is found in the plasma as full length and truncated forms. The latter is more active and generated by cleavage at a Furin-like site. Endothelial lipase (EL) binds heparin sulfate proteoglycans on cell surfaces and catalyzes the hydrolysis of HDL phospholipids. EL activity is regulated by two endogenous inhibitors, ANGPTL3 and ANGPTL4, and by PCs that inactivate EL through cleavage releasing the N-terminal catalytic and C-terminal lipid-binding domains. Herein, because Furin and PC5/6 complete KOs are lethal, we used mice lacking Furin or PC5/6 specifically in hepatocytes (hKO) or mice completely lacking PACE4. In primary hepatocytes, ANGPTL3 was processed into a shorter form of ANGPTL3 intracellularly by Furin only, and extracellularly mainly by PACE4. In vivo, the absence of Furin in hepatocytes reduced by ∼50% the circulating levels of cleaved ANGPTL3, while the lack of PACE4 had only a minor effect. Analysis of the EL processing in primary hepatocytes and in vivo revealed that it is mostly cleaved by Furin. However, the lack of Furin or PC5/6 in hepatocytes and complete PACE4 KO did not appreciably modify plasma HDL levels or EL activity. Thus, inhibition of Furin in liver would not be expected to modify the plasma lipid profiles.
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Loss of Endothelial Furin Leads to Cardiac Malformation and Early Postnatal Death
Molecular and Cellular Biology, 2012Co-Authors: Rachid Essalmani, Nabil G. Seidah, Anton J M Roebroek, Dorota Szumska, John W.m. Creemers, Pedro D'orléans-juste, Shoumo Bhattacharya, Annik PratAbstract:In mammals, seven proprotein convertases (PCs) cleave secretory proteins after basic residues, and four of them are called Furin-like PCs: Furin, PC5, PACE4, and PC7. In vitro, they share many substrates. However, Furin is essential during development since deficient embryos die at embryonic day 11 and exhibit multiple developmental defects, particularly defects related to the function of endothelial cells. To define the role of Furin in endothelial cells, an endothelial cell-specific knockout (ecKO) of the Furin gene was generated. Newborns die shortly after birth, indicating that Furin is essential in these cells. Magnetic resonance imaging revealed that ecKO embryos exhibit ventricular septal defects (VSD) and/or valve malformations. In addition, primary cultures of wild-type and ecKO lung endothelial cells revealed that ecKO cells are unable to grow. Growth was efficiently rescued by extracellular soluble Furin. Analysis of the processing of precursors of endothelin-1 (ET-1), adrenomedullin (Adm), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein 4 (BMP4) confirmed that ET-1, Adm, and TGF-β1 are in vivo substrates of endothelial Furin. Mature ET-1 and BMP4 forms were reduced by ~90% in ecKO purified endothelial cells from lungs.
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Heparin enhances the Furin cleavage of HIV‐1 gp160 peptides
FEBS Letters, 2007Co-Authors: Antonella Pasquato, Nabil G. Seidah, Monica Dettin, A. Basak, Roberta Gambaretto, L. Tonin, C. Di BelloAbstract:Infectious HIV-1 requires gp160 cleavage by Furin at the REKR511↓ motif (site1) into the gp120/gp41 complex, whereas the KAKR503 (site2) sequence remains uncleaved. We synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro Furin processing. We found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro Furin substrates, the present extended sequences require heparin for optimal processing. Our data support the hypothesis of a direct binding of heparin with site1 and site2, allowing selective exposure/accessibility of the REKR sequence, which is only then optimally cleaved by Furin.
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Inactivation of Furin by its naturally occurring inhibitor pro-Furin abolish breast cancer cells malignant phenotypes and tumorigenecity in mice.
Cancer Research, 2006Co-Authors: Nathalie Scamuffa, Nabil G. Seidah, Ajoy Basak, Fabien Calvo, Abdel-majid KhatibAbstract:5269 Maturation of various cancer-related substrates by the ubiquitous proprotein convertase (PC) Furin was reported to be a crucial step in various processes of tumor progression and metastasis. Like its substrates, the Furin is also synthesized as inactive proenzyme and is auto-catalytically activated. Following signal sequence removal, Furin undergo auto-proteolytic cleavage of its prosegment domain (pro-Furin). The latter remains associated with the enzyme and functions as a potent auto-inhibitor. After this step, the inactive complex transits to late Trans-Golgi Network where a second auto-proteolytic cleavage of the prosegment occurs and activates the Furin. In the present study we sought to determine whether the pro-Furin mediated-loss of Furin-activity might affect the malignant phenotypes of breast cancer cells. Overexpression of pro-Furin cDNA in breast cancer cells in a stable manner resulted in processing blockade of various PC substrates including IGF-1 receptor, PDGF-A, PDGF-B and VEGF-C. Similarly, using the reverse transcription-polymerase chain reaction, we found that various genes involved in tumorigenesis such as MMPs were significantly downregulated in pro-Furin overexpressing cells. In vitro and in vivo comparative studies revealed that these cells exhibited a reduced ability to proliferate and when subcutaneously inoculated in nude mice they induced delayed tumors with reduced size and incidence. Finally, using pro-Furin derived synthetic peptides with various sequence lengths revealed that only the full length pro-Furin peptide was able to mediate an inhibitory effect \. on breast cancer cells growth. These findings suggest that Furin inhibition by its naturally occurring inhibitor pro-Furin may be a useful strategy in breast cancer therapy.
Michele Bernasconi - One of the best experts on this subject based on the ideXlab platform.
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the proprotein convertase Furin in tumour progression
International Journal of Cancer, 2017Co-Authors: Patricia Jaaks, Michele BernasconiAbstract:Proprotein convertases are proteases that have been implicated in the activation of a wide variety of proteins. These proteins are generally synthesised as precursor proteins and require limited proteolysis for conversion into their mature bioactive counterparts. Many of these proteins, including metalloproteases, growth factors and their receptors or adhesion molecules, have been shown to facilitate tumour formation and progression. Hence, this review will focus on the proprotein convertase Furin and its role in cancer. The expression of Furin has been confirmed in a large spectrum of cancers such as head and neck squamous cell carcinoma, breast cancer and rhabdomyosarcoma. Functional studies modulating Furin activity uncovered its importance for the processing of many cancer-related substrates and strongly indicate that high Furin activity promotes the malignant phenotype of cancer cells. In this review, we summarise the expression and function of Furin in different cancer types, discuss its role in processing cancer-related proproteins and give examples of potential therapeutic approaches that take advantage of the proteolytic activity of Furin in cancer cells.
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The proprotein convertase Furin is required to maintain viability of alveolar rhabdomyosarcoma cells
Oncotarget, 2016Co-Authors: Patricia Jaaks, Beat W. Schäfer, Gianmarco Meier, Nagjie Alijaj, Eva Brack, Peter K. Bode, Ewa Koscielniak, Marco Wachtel, Michele BernasconiAbstract:// Patricia Jaaks 1 , Gianmarco Meier 1 , Nagjie Alijaj 1 , Eva Brack 1 , Peter Bode 2 , Ewa Koscielniak 3 , Marco Wachtel 1 , Beat W. Schafer 1 , Michele Bernasconi 1 1 Department of Oncology and Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland 2 Department of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland 3 Department of Oncology/Hematology/Immunology, Olgahospital, Klinikum Stuttgart, Stuttgart, Germany Correspondence to: Michele Bernasconi, email: michele.bernasconi@kispi.uzh.ch Keywords: Furin, proprotein convertases, rhabdomyosarcoma, apoptosis, IGF1R Received: February 10, 2016 Accepted: August 09, 2016 Published: August 27, 2016 ABSTRACT Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Success of current therapies is still limited and outcome is particularly poor for metastatic alveolar rhabdomyosarcoma (aRMS). We previously identified the proprotein convertase Furin as potential target for specific drug delivery with RMS-homing peptides. Furin is a protease that converts inactive precursor proteins into bioactive proteins and peptides. In this study, we investigate the biological role of Furin in aRMS progression in vitro and in vivo . Furin expression was confirmed in over 86% RMS biopsies in a tissue microarray (n=89). Inducible Furin silencing in vitro led to significant impairment of cell viability and proliferation in all investigated aRMS cell lines, but not in MRC5 fibroblasts. Furthermore, the aRMS cell lines Rh3 and Rh4 revealed to be very sensitive to Furin silencing, undergoing caspase-dependent cell death. Notably, Furin silencing in vivo led to complete remission of established Rh4 tumors and to delayed growth in Rh30 tumors. Taken together, these findings identify Furin as an important factor for aRMS progression and survival. Thus, we propose Furin as a novel therapeutic target for treatment of aRMS.
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The Proprotein Convertase Furin Contributes to Rhabdomyosarcoma Malignancy by Promoting Vascularization, Migration and Invasion.
PLOS ONE, 2016Co-Authors: Patricia Jaaks, Valentina D'alessandro, Nicole Grob, Sina Büel, Katarina Hajdin, Beat W. Schäfer, Michele BernasconiAbstract:The proprotein convertase (PC) Furin cleaves precursor proteins, an important step in the activation of many cancer-associated proteins. Substrates of Furin and Furin-like PCs play a role in proliferation, metastasis and invasion. Some of them are involved in the progression of the pediatric soft tissue sarcoma rhabdomyosarcoma (RMS). In this study, we show that PCs, and in particular Furin, are expressed in RMS cell lines. To investigate the functional role of Furin, we generated RMS cell lines with modulated Furin activity. Silencing or stable inhibition of Furin delayed tumor growth in Rh30 and RD xenografts in vivo, and was correlated with lower microvessel density. Reduced Furin activity also decreased migration and invasion abilities in vitro, and inhibition of Furin in RMS cells diminished processing of IGF1R, VEGF-C, PDGF-B and MT1-MMP, leading to lower levels of mature proteins. Furthermore, we found that Furin activity is required for proper IGF signaling in RMS cells, as Furin silencing resulted in reduced phosphorylation of Akt upon IGF1 stimulation. Taken together, our results suggest that Furin plays an important role in the malignant phenotype of RMS cells by activating proteins involved in tumor growth and vascularization, metastasis and invasion.
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Abstract 3571: Furin activity: A driver of rhabdomyosarcoma progression
Cancer Research, 2015Co-Authors: Patricia Jaaks, Beat W. Schäfer, Gianmarco Meier, Michele BernasconiAbstract:Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The prognosis and survival rate is often very poor and therefore new therapy approaches are required. Thus, we focus on 1) employing RMS-specific peptides for targeted delivery of chemotherapeutics to the tumor site and 2) identifying novel therapeutic targets. In a previous study we discovered that RMS-homing peptides bind preferably to the proprotein convertase Furin. Initial investigations confirmed a high expression of Furin throughout different pediatric soft tissue sarcoma types and hinted that Furin promotes the tumorigenic phenotype of RMS cells in vitro as well as in corresponding xenografts in vivo. Here we present a novel approach of tetracycline-induced shRNA-based silencing of Furin in vitro and in vivo in order to investigate in depth the role of Furin in RMS progression. Furin depletion was confirmed at mRNA, protein and activity level and led to impaired maturation Furin substrates insulin like growth factor 1 receptor (IGF1R) and transforming growth factor beta 1 (TGFβ-1). We found that loss of Furin activity suppresses the malignant phenotype of Rh30 cells by decreasing proliferation and affecting formation of colonies. Furthermore, we observed increased caspase 3/7 activity and enrichment of nucleosomes in the cytoplasm upon Furin depletion, thus hinting initiation of apoptotic processes. Induction of Furin silencing in RMS xenografts in NOD/SCID mice delayed tumour growth, indicating a crucial role of Furin in early phases of tumour growth. Taken together, our data underscore the importance of Furin for RMS progression and therefore targeting the activity of Furin represents a promising tool for treatment of RMS. Citation Format: Patricia AIM Jaaks, Gianmarco Meier, Beat W. Schafer, Michele Bernasconi. Furin activity: A driver of rhabdomyosarcoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3571. doi:10.1158/1538-7445.AM2015-3571
