Department of Tumor Biology, Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, 84505, Bratislava, Slovakia.
Institute of Pharmacology and Toxicology, RWTH Aachen University, Wendlingweg 2, 52074, Aachen, Germany.
Br J Cancer. 2020 May;122(11):1590-1603. doi: 10.1038/s41416-020-0804-z. Epub 2020 Mar 25.
Carbonic 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.
We 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.
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. 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.
Our 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.
碳酸酐酶 IX（CA IX）是一种缺氧诱导的酶，可调节肿瘤 pH 值并促进细胞迁移/侵袭。它主要作为跨膜细胞表面蛋白表达，但它的胞外结构域可以通过 ADAM17 被切割到细胞外空间。本研究旨在阐明 CA IX 脱落对癌细胞的影响。
我们通过从茎部区域缺失氨基酸 393-402 生成了非脱落 CA IX 突变体，并使用基于免疫检测、共聚焦显微镜、体外实时细胞监测和异种移植 NMRI 和 C57BL/6J 雌性小鼠体内肿瘤细胞接种的一系列测定来研究其与全长、脱落能力 CA IX 的表型效应。
我们证明，脱落的损伤不会改变 CA IX 与 ADAM17 结合、内化、形成寡聚体和调节 pH 的能力，但会诱导细胞外蛋白质组中促进癌症的变化。此外，它还会影响表达非脱落变体的细胞的内在特性，表现为其迁移、生成原发性肿瘤和在肺部形成转移性病变的能力增强。
我们的结果表明，胞外结构域的脱落控制了细胞相关 CA IX 的促肿瘤发生和促转移作用，并表明在开发 CA IX 靶向治疗策略时应考虑到这一现象。
