Abstract
BackgroundCarbonic 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.MethodsPresence 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.ResultsWe 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.ConclusionsThese 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.
Highlights
Carbonic anhydrase carbonic anhydrase IX protein (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
To get insight into this phenomenon, we investigated the relationship between Carbonic anhydrase IX (CA IX) expression and the response of cancer cells to cytotoxic treatment
Flow cytometry revealed that approximately half of the normoxic CGL3 cells show the cell surface expression of CA IX and that labeling with the CA IX-specific antibody M75 allows for an easy gating and separate analysis of the CA IXpositive and CA IX-negative subpopulations growing together in the same culture dish
Summary
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. Carbonic anhydrase IX (CA IX) belongs to a carbonic anhydrase family of enzymes that use a zinc-activated hydroxide mechanism to catalyze the reversible conversion of carbon dioxide to carbonic acid in the net reaction CO2 + H2O ↔ HCO3− + H+ [1, 2] Via this catalytic activity, carbonic anhydrases either supply bicarbonate for biosynthetic reactions and ion transport across membranes or consume produced/transported bicarbonate. CA IX is closely associated with a broad range of tumors that either suffer from hypoxia or contain inactive pVHL [5] This expression pattern is principally determined by a strong HIF-1-mediated transcriptional activation of the CA9 gene, which contains an HRE element localized on the negative DNA strand immediately upstream of the transcription start site [5]
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