title: Apoptosis-induced ectodomain shedding of hypoxia-regulated carbonic anhydrase IX from tumor cells: a double-edged response to chemotherapy.
abstract: BACKGROUND
  Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin.
  
  
  METHODS
  Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay.
  
  
  RESULTS
  We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness.
  
  
  CONCLUSIONS
  These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, 845 05, Bratislava, Slovak Republic.|Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.
authors.forename: Ivana
authors.initials: I
authors.lastname: Vidlickova
authors.affiliation: Cellular and Molecular Biology Unit, Gembloux Agro-Bio Tech, University of Liege, Liege, Belgium.
authors.forename: Franck
authors.initials: F
authors.lastname: Dequiedt
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, 845 05, Bratislava, Slovak Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Jelenska
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, 845 05, Bratislava, Slovak Republic.
authors.forename: Olga
authors.initials: O
authors.lastname: Sedlakova
authors.affiliation: Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Pastorek
authors.affiliation: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Stuchlik
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, 845 05, Bratislava, Slovak Republic.
authors.forename: Jaromir
authors.initials: J
authors.lastname: Pastorek
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, 845 05, Bratislava, Slovak Republic.
authors.forename: Miriam
authors.initials: M
authors.lastname: Zatovicova
authors.affiliation: Department of Molecular Medicine, Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, 845 05, Bratislava, Slovak Republic. silvia.pastorekova@savba.sk.|Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic. silvia.pastorekova@savba.sk.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pastorekova
mesh_terms: D000072198:ADAM17 Protein / Q000235:genetics* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000008:administration & dosage; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D000071231:Carbonic Anhydrase IX / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism; D015687:Cell Hypoxia / Q000235:genetics; D003513:Cycloheximide / Q000008:administration & dosage; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008297:Male; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: D000911:Antibodies, Monoclonal; D003513:Cycloheximide; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D000071231:Carbonic Anhydrase IX
keywords: Apoptosis; Carbonic anhydrase IX; Chemotherapy; Ectodomain; Hypoxia; Metalloproteinase; Shedding