Abstract
BackgroundCarbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells.MethodsWe generated a non-shed CA IX mutant by deletion of amino acids 393–402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice.ResultsWe demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs.ConclusionsOur results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.
Highlights
Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/ invasion
Deletions in the CA IX stalk region define the ADAM17 cleavage region and generate non-shed CA IX variant We previously demonstrated that CA IX shedding depends on the presence of ADAM17.7 In this study, we first wanted to obtain biochemical evidence that CA IX is a direct substrate of ADAM17
In order to tackle the role of cell-associated CA IX in the absence of its ectodomain cleavage, we used an approach based on construction and ectopic expression of the non-shed (NS) CA IX mutant
Summary
Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/ invasion. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome It affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. Ectodomain cleavage ( called shedding) is an important regulatory phenomenon that controls abundance and/or function of cell-membrane-bound proteins as well as molecular composition of the extracellular microenvironment and thereby influences both cell’s phenotype and intercellular signalling Deregulation of this process leading to imbalance between cell-associated and soluble variants of certain proteins has been associated with diverse diseases including infections, inflammatory disorders and cancer. Prototypic member of this family ADAM17 (initially named TNFα converting enzyme, TACE) is currently known to cleave around 80 biologically relevant transmembrane proteins.[2,3]
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