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

Abstract

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.

Fig. 1.

Analysis of EMT induction in human prostate epithelial cell lines WPE and PNT1a. Cells were treated with TGF-β or EtDO-P4, and analyzed for EMT induction as described in Materials and Methods and SI Materials and Methods. (I) Cell morphology changes. (Top) WPE cells. (Bottom) PNT1a cells. (Left) Control. (Center) TGF-β treatment. (Right) EtDO-P4 treatment. (II) Analysis of epithelial vs. mesenchymal cell markers by SDS/PAGE and Western blot. (A, C, and E) WPE cells. (B, D, and F) PNT1a cells. Samples containing 10 μg protein were used with γ-tubulin as loading control. (A and B) Experiments were performed in triplicate, and representative results are shown. (C–F) Signal intensities were normalized, and values are shown as relative intensity (mean ± SD) for Ecad (C and D), Ncad (E), and vimentin (F). n.s.: not significant; *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001.

Fig. 2.

Expression of total FN and onfFN after treatment with TGF-β or EtDO-P4. Cells were treated and cell lysates were prepared as described in SI Materials and Methods, and samples were subjected to SDS/PAGE and Western blot as described in Fig. 1. Total FN and onfFN were detected with mAb EP5 (A and C) and mAb FDC6 (B and D), respectively. Human plasma FN (0.5 μg) and HUH-7 cell lysate (10 μg) were used as positive controls. Western blot results were analyzed as described in Fig.1, and mean ± SD is shown. n.s.: not significant; **P ≤ 0.005; ***P ≤ 0.001.

Fig. 3.

Western blot analysis for GalNAc-Ts and RT-qPCR analysis for GalNAc-Ts and FN IIICS domain. WPE cells were transfected with control siRNA or siRNA for T3/T6, and then treated with TGF-β as described in Materials and Methods. Selectivity of the siRNA was evaluated by Western blot using specific mAbs. (I) Representative Western blot for T2, T3 and T6 from triplicate experiments (A) and mean ± SD (B–D) are shown. (II) Results from RT-qPCR for GalNAc-T2, T3, T6, and FN IIICS domain from triplicate experiments are normalized with values for actin and mean ± SD are shown. RQ, relative quantity. n.s.: not significant . *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001.

Fig. 4.

Effect of knockdown of GalNAc-T3/T6 on TGF-β–induced EMT, assessed by cell morphology. WPE and PNT1a cells were transfected with a mixture of siRNA duplexes for human GalNAc-T3 and T6 to obtain double knockdown cells, or with negative siRNA for control cells, and then treated with TGF-β as described in SI Materials and Methods. Representative photos from triplicate experiments are shown.

Fig. 5.

Effect of knockdown of GalNAc-T3/T6 on TGF-β–induced EMT in WPE cells, assessed by expression of epithelial and mesenchymal cell markers. WPE cells were transfected with siRNA duplexes or negative siRNA, and treated with TGF-β, as in Fig. 4. Cell lysates were prepared for Western blot analysis as described in SI Materials and Methods. (A) Representative results from quadruplicate experiments. (C–F) Relative expression levels normalized with loading control were calculated, and shown as mean ± SD; n.s.: not significant; *P ≤ 0.05; **P ≤ 0.005. (B) Total FN and onfFN secreted in the culture supernatants was also analyzed with WPE cells. Representative results from triplicate experiments are shown.

Fig. 6.

Effect of knockdown of GalNAc-T3/T6 on TGF-β–induced EMT, assessed by cell motility. WPE and PNT1a cells were transfected with siRNA duplexes or negative siRNA, and treated with TGF-β, as in Fig. 3. Cell motility was analyzed by phagokinetic assay, as described in SI Materials and Methods. (A–C) No TGF-β treatment. (D–F) TGF-β treatment. (B and E) Negative control siRNA. (C and F) GalNAc-T3/T6 siRNA mixture. (G) Tracks from 50 individual cells were measured and quantified as described in SI Materials and Methods, and mean ± SD is shown. ***P ≤ 0.001.