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 ␣ 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 ␣ 3 and ␤ 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. Molecular & Cellular Proteomics 13: 10.1074/mcp.M114.038505, 3126-3137, 2014.As solid tumors grow, those areas distant from the existing blood vessels can become chronically or intermittently deprived of sufficient oxygen. These hypoxic conditions place tremendous pressure on tumor cells and drive the development of increasingly malignant and metastatic phenotypes (1, 2). As metastases are responsible for more than 90% of human cancer-related deaths, much research has aimed to define the underlying molecular mechanisms of the tumor cell response to a hypoxic microenvironment (3-5). However, despite the evident clinical relevance of metastasis, the full complexity of the process remains incompletely understood. An emerging area of interest is the importance of the carbohydrate structures in tumor cells, which have been linked to control of protein folding and stability, cell-cell recognition, adhesion, invasion, and metastatic potentials (6 -11). The post-translational enzymatic addition of glycans (glycosylation) to proteins is a potent modulator of the functions of many receptors involved in cell growth, adhesion, and signal transduction (11)(12)(13)(14) and is commonly seen in both in vitro and in vivo cancer models (15, 16). Furthermore, a growing body of studies has shown a clear correlation betw...

Inhibition of pancreatic tumoral cells by snake venom disintegrins

“…The laminin from Engelbreth-Holm-Swarm (EHS) tumor used in this study is usually considered laminin-1 (Patarroyo et al, 2002). Integrin α 3 β 1 was originally identified as a promiscuos receptor for a range of ligands including collagen, fibronectin, entactin, laminin-5, thrombospondin-1 and the laminin used in this study, lamini-1 (Ren et al, 2014). Most integrins bind to several ECM molecules, and most ECM bind to more than one integrin heterodimer.…”

Glycobiology Modifications in Intratumoral Hypoxia: The Breathless Side of Glycans Interaction

“…In human epithelial carcinoma (A431), Ren et al reported that hypoxia-induced alterations to the glycosylation of ITGA3 prevented its efficient translocation to the plasma membran. This resulted in an enhanced invasive competence to cancer cells [97]. In another indirect correlation between PGs, integrins, and hypoxia, You et al showed that pericyte expression of NG2 (neuron glia antigen-2) is critical for its interaction with endothelial cells in a NG2 knockdown melanoma (B16F10 lineage) model.…”

“…Some studies have reported molecular changes promoted by the hypoxia microenvironment, but did not elucidate the functional consequences promoted by glycoconjugates alterations in neoplastic cells. However, some studies associated these modifications with cancer cell proliferation, migration, and survival, indicating that such glycoconjugates may be used as targets for further therapeutic strategies for cancer treatment [97-99]. …”

“…Mutagenesis studies indicated that lower levels of ITGA3 on the cell surface, such as hypoxia-induced levels, increase the invasiveness potential of cancer cells in vitro by blocking ITGA3’s interaction with extracellular ligands (Fig. 4) [97]. …”

“…A study performed by Ren et al evaluated the effect of hypoxia on the integrin family in A431 epidermoid carcinoma cells [97]. Expressed glycoproteins were identified and quantified by iTRAQ (isobaric tags for relative and absolute quantitation) and liquid chromatography-mass spectrometry analysis.…”