Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431

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 focuse...

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Main Authors: Ren, Yan, Hao, Piliang, Law, Sai-Kit Alex, Sze, Siu Kwan
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2015
Online Access:https://hdl.handle.net/10356/80751
http://hdl.handle.net/10220/38979
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spelling sg-ntu-dr.10356-807512023-02-28T16:59:15Z Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431 Ren, Yan Hao, Piliang Law, Sai-Kit Alex Sze, Siu Kwan School of Biological Sciences 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. NMRC (Natl Medical Research Council, S’pore) Accepted version 2015-12-07T06:57:13Z 2019-12-06T13:58:09Z 2015-12-07T06:57:13Z 2019-12-06T13:58:09Z 2014 Journal Article Ren, Y., Hao, P., Law, S.-K. A., & Sze, S. K. (2014). Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431. Molecular & Cellular Proteomics, 13(11), 3126-3137. https://hdl.handle.net/10356/80751 http://hdl.handle.net/10220/38979 10.1074/mcp.M114.038505 25078904 en Molecular & Cellular Proteomics © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. This is the author created version of a work that has been peer reviewed and accepted for publication by Molecular & Cellular Proteomics, The American Society for Biochemistry and Molecular Biology, Inc. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1074/mcp.M114.038505]. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
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collection DR-NTU
language English
description 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.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Ren, Yan
Hao, Piliang
Law, Sai-Kit Alex
Sze, Siu Kwan
format Article
author Ren, Yan
Hao, Piliang
Law, Sai-Kit Alex
Sze, Siu Kwan
spellingShingle Ren, Yan
Hao, Piliang
Law, Sai-Kit Alex
Sze, Siu Kwan
Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431
author_sort Ren, Yan
title Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431
title_short Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431
title_full Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431
title_fullStr Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431
title_full_unstemmed Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431
title_sort hypoxia-induced changes to integrin α 3 glycosylation facilitate invasion in epidermoid carcinoma cell line a431
publishDate 2015
url https://hdl.handle.net/10356/80751
http://hdl.handle.net/10220/38979
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