Abstract
Anoikis is a fundamental cellular process for maintaining tissue homeostasis. Resistance to anoikis is a hallmark of oncogenic epithelial–mesenchymal transition and is a pre-requisite for metastasis. Previous studies have revealed that the heavily glycosylated mucin protein MUC1, which is overexpressed in all types of epithelial cancer cells, prevents anoikis initiation in response to loss of adhesion. This effect of MUC1 is largely attributed to its extracellular domain that provides cell surface anoikis-initiating molecules with a ‘homing’ microenvironment. The present study investigated the influence of O-glycosylation on MUC1 extracellular domain on MUC1-mediated cell resistance to anoikis. It shows that stable suppression of the Core 1Gal-transferase (C1GT) by shRNA substantially reduces O-glycosylation in MUC1-positively transfected human colon cancer HCT116 cells and in high MUC1-expressing SW620 cells. Suppression of C1GT significantly increased anoikis of the MUC1-positive, but not MUC1-negative, cells in response to suspended culture. This effect was shown to be associated with increased ligand accessibility to cell surface anoikis-initiating molecules such as E-cadherin, integrinβ1 and Fas. These results indicate that the extensive O-glycosylation on MUC1 extracellular domain contributes to MUC1-mediated cell resistance to anoikis by facilitating MUC1-mediated prohibition of activation of the cell surface anoikis-initiating molecules in response to loss of cell adhesion. This provides insight into the molecular mechanism of anoikis regulation and highlights the importance of cellular glycosylation in cancer progression and metastasis.
Highlights
Anoikis is a special apoptotic process that occurs in cells in response to loss of adhesion to the extracellular matrix.[1,2] It is a fundamental cellular process for maintaining tissue homeostasis by removing displaced epithelial/endothelial cells and preventing them from seeding to inappropriate sites.Anoikis initiation starts from the cell surface through activation of the cell surface anoikis-initiating molecules such as integrins and death receptors
Core 1Gal-transferase (C1GT) is a key glycosyltransferase in the biosynthesis of O-linked mucin type glycans.[17]
We have shown that partial inhibition of O-glycosylation by stable suppression of C1GT expression significantly increased anoikis of MUC1-positive, but not MUC1-negative, cells in response to suspended culture
Summary
Anoikis is a special apoptotic process that occurs in cells in response to loss of adhesion to the extracellular matrix.[1,2] It is a fundamental cellular process for maintaining tissue homeostasis by removing displaced epithelial/endothelial cells and preventing them from seeding to inappropriate sites. Interaction of Fas-L with Fas on the cell surface is known to induce anoikis/apoptosis through activation of caspase-8.4,25 It was found that introduction of exogenous Fas-L significantly increased the caspase-8 activity of the MUC1-negative (Neo) cells but had little influence on the MUC1-positive (F3) cells, again supporting the role of MUC1 expression on inhibition of Fas-L-Fas interaction-induced anoikis as previously reported.[16] Suppression of C1GT in F3 cells (E7 and B7) resulted in significant increase of caspase-8 activity in cell response to anoikis culture, such an effect was not observed in the control shRNA transfected cells (F2 and C8; Figure 5) These results again support a role of MUC1 O-glycosylation in MUC1-mediated anoikis inhibition by participating in preventing anoikis-initiating molecule interactions with their ligands on cell surface.
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