Involvement of O-glycosylation defining oncofetal fibronectin in epithelial-mesenchymal transition process

TY - JOUR

T1 - Involvement of O-glycosylation defining oncofetal fibronectin in epithelial-mesenchymal transition process

AU - Freire-de-Lima, Leonardo

AU - Gelfenbeyn, Kirill

AU - Ding, Yao

AU - Mandel, Ulla

AU - Clausen, Henrik

AU - Handa, Kazuko

AU - Hakomori, Sen-Itiroh

PY - 2011/10/25

Y1 - 2011/10/25

N2 - The process termed "epithelial-mesenchymal transition" (EMT) was originally discovered in ontogenic development, and has been shown to be one of the key steps in tumor cell progression and metastasis. Recently,we showed that the expression of some glycosphingolipids (GSLs) is down-regulated during EMT in human and mouse cell lines. Here, we demonstrate the involvement of GalNAc-type (or mucin-type) O-glycosylation in EMT process, induced with transforming growth factor β (TGF-β) in human prostate epithelial cell lines. We found that: (i) TGF-β treatment caused up-regulation of oncofetal fibronectin (onfFN), which is defined by mAb FDC6, and expressed in cancer or fetal cells/tissues, but not in normal adult cells/tissues. The reactivity of mAb FDC6 requires the addition of an O-glycan at a specific threonine, inside the type III homology connective segment (IIICS) domain of FN. (ii) This change is associated with typical EMT characteristics; i.e., change from epithelial to fibroblastic morphology, enhanced cell motility, decreased expression of a typical epithelial cell marker, E-cadherin, and enhanced expression of mesenchymal markers. (iii) TGF-β treatment up-regulated mRNA level of FN containing the IIICS domain and GalNAc-T activity for the IIICS domain peptide substrate containing the FDC6 onfFN epitope. (iv) Knockdown of GalNAc-T6 and T3 inhibited TGF-β-induced up-regulation of onfFN and EMT process. (v) Involvement of GSLs was not detectable with the EMT process in these cell lines. These findings indicate the important functional role of expression of onfFN, defined by site-specific O-glycosylation at IIICS domain, in the EMT process.

AB - The process termed "epithelial-mesenchymal transition" (EMT) was originally discovered in ontogenic development, and has been shown to be one of the key steps in tumor cell progression and metastasis. Recently,we showed that the expression of some glycosphingolipids (GSLs) is down-regulated during EMT in human and mouse cell lines. Here, we demonstrate the involvement of GalNAc-type (or mucin-type) O-glycosylation in EMT process, induced with transforming growth factor β (TGF-β) in human prostate epithelial cell lines. We found that: (i) TGF-β treatment caused up-regulation of oncofetal fibronectin (onfFN), which is defined by mAb FDC6, and expressed in cancer or fetal cells/tissues, but not in normal adult cells/tissues. The reactivity of mAb FDC6 requires the addition of an O-glycan at a specific threonine, inside the type III homology connective segment (IIICS) domain of FN. (ii) This change is associated with typical EMT characteristics; i.e., change from epithelial to fibroblastic morphology, enhanced cell motility, decreased expression of a typical epithelial cell marker, E-cadherin, and enhanced expression of mesenchymal markers. (iii) TGF-β treatment up-regulated mRNA level of FN containing the IIICS domain and GalNAc-T activity for the IIICS domain peptide substrate containing the FDC6 onfFN epitope. (iv) Knockdown of GalNAc-T6 and T3 inhibited TGF-β-induced up-regulation of onfFN and EMT process. (v) Involvement of GSLs was not detectable with the EMT process in these cell lines. These findings indicate the important functional role of expression of onfFN, defined by site-specific O-glycosylation at IIICS domain, in the EMT process.

KW - Animals

KW - Antibodies, Monoclonal

KW - Blotting, Western

KW - Cadherins

KW - Cell Line

KW - DNA Primers

KW - Epithelial Cells

KW - Epithelial-Mesenchymal Transition

KW - Fibronectins

KW - Gene Expression Regulation, Neoplastic

KW - Gene Knockdown Techniques

KW - Glycosylation

KW - Humans

KW - Mice

KW - N-Acetylgalactosaminyltransferases

KW - RNA, Messenger

KW - RNA, Small Interfering

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Transforming Growth Factor beta

U2 - 10.1073/pnas.1115191108

DO - 10.1073/pnas.1115191108

M3 - Journal article

C2 - 22006308

SN - 0027-8424

VL - 108

SP - 17690

EP - 17695

JO - Proceedings of the National Academy of Sciences USA (PNAS)

JF - Proceedings of the National Academy of Sciences USA (PNAS)

IS - 43

ER -