title: Hypoxia-induced changes to integrin α 3 glycosylation facilitate invasion in epidermoid carcinoma cell line A431.
abstract: Hypoxia is a critical microenvironmental factor that drives cancer progression through angiogenesis and metastasis. Glycoproteins, especially those on the plasma membrane, orchestrate this process; however, questions remain regarding hypoxia-perturbed protein glycosylation in cancer cells. We focused on the effects of hypoxia on the integrin family of glycoproteins, which are central to the cellular processes of attachment and migration and have been linked with cancer in humans. We employed electrostatic repulsion hydrophilic interaction chromatography coupled with iTRAQ labeling and LC-MS/MS to identify and quantify glycoproteins expressed in A431. The results revealed that independent of the protein-level change, N-glycosylation modifications of integrin α 3 (ITGA3) were inhibited by hypoxia, unlike in other integrin subunits. A combination of Western blot, flow cytometry, and cell staining assays showed that hypoxia-induced alterations to the glycosylation of ITGA3 prevented its efficient translocation to the plasma membrane. Mutagenesis studies demonstrated that simultaneous mutation of glycosites 6 and 7 of ITGA3 prevented its accumulation at the K562 cell surface, which blocked integrin α 3 and β 1 heterodimer formation and thus abolished ITGA3's interaction with extracellular ligands. By generating A431 cells stably expressing ITGA3 mutated at glycosites 6 and 7, we showed that lower levels of ITGA3 on the cell surface, as induced by hypoxia, conferred an increased invasive ability to cancer cells in vitro under hypoxic conditions. Taken together, these results revealed that ITGA3 translocation to the plasma membrane suppressed by hypoxia through inhibition of glycosylation facilitated cell invasion in A431.
authors: From the ‡School of Biological Sciences, Nanyang Technological University, 60 Nanyang Dr., Singapore 637551, Singapore.
authors: Yan
authors: Y
authors: Ren
authors: From the ‡School of Biological Sciences, Nanyang Technological University, 60 Nanyang Dr., Singapore 637551, Singapore.
authors: Piliang
authors: P
authors: Hao
authors: From the ‡School of Biological Sciences, Nanyang Technological University, 60 Nanyang Dr., Singapore 637551, Singapore.
authors: S K Alex
authors: SK
authors: Law
authors: From the ‡School of Biological Sciences, Nanyang Technological University, 60 Nanyang Dr., Singapore 637551, Singapore. sksze@ntu.edu.sg.
authors: Siu Kwan
authors: SK
authors: Sze
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000473:pathology*; D002448:Cell Adhesion; D015687:Cell Hypoxia / Q000502:physiology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002853:Chromatography, Liquid; D006023:Glycoproteins / Q000032:analysis / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D039422:Integrin alpha3 / Q000235:genetics / Q000378:metabolism*; D019012:Integrin beta1 / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009361:Neoplasm Invasiveness / Q000473:pathology*; D021381:Protein Transport / Q000502:physiology; D034622:RNA Interference; D034741:RNA, Small Interfering; D053719:Tandem Mass Spectrometry
chemical_list: D006023:Glycoproteins; C000599264:ITGA3 protein, human; D039422:Integrin alpha3; D019012:Integrin beta1; D034741:RNA, Small Interfering