CRISPR-screen identifies ZIP9 and dysregulated Zn2+ homeostasis as a cause of cancer-associated changes in glycosylation

Troels Boldt Rømer, Fawzi Khoder-Agha, Mikkel Koed Møller Aasted, Noortje de Haan, Sabrina Horn, August Dylander, Tao Zhang, Emil Marek Heymans Pallesen, Sally Dabelsteen, Manfred Wuhrer, Christine Flodgaard Høgsbro, Emil Aagaard Thomsen, Jacob Giehm Mikkelsen, Hans H Wandall


In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are up-regulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear. To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn. We show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in up-regulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as down-regulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.

StatusE-pub ahead of print - 16 jan. 2023


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