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I-branching N-acetylglucosaminyltransferase regulates prostate cancer invasiveness by enhancing α5β1 integrin signaling.

Mikami J, Tobisawa Y, Yoneyama T, Hatakeyama S, Mori K, Hashimoto Y, Koie T, Ohyama C, Fukuda M - Cancer Sci. (2016)

Bottom Line: GCNT2-positive cells were significantly lesser in organ-confined disease than in that with extra-capsular extensions, and GCNT2-negative tumors were associated with significantly better prostate-specific antigen-free survival compared with GCNT2-positive tumors.Subsequent functional studies revealed that knockdown of GCNT2 expression in PCa cell lines significantly inhibited cell migration and invasion.In conclusion, GCNT2 expression is closely associated with invasive potential of PCa.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

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I‐antigen regulates cell spread and α5β1 integrin–fibronectin interactions that are mediated by protein kinase B (AKT) phosphorylation in DU145NC and DU145GCNT2KD4 cells. Cell adhesion and spreading on fibronectin (FN) was examined by harvesting cells after a 30 min of culture on fibronectin‐coated 96‐well plates (a). DU145NC and DU145GCNT2KD4 cells showed no significant differences in adhesion potential. (b) Cells were harvested from fibronectin‐coated glass slides after 30 min of incubation, and spreading cells were visualized using crystal violet solution. Percentages of spreading cells were significantly lower in DU145GCNT2KD4 cells than in DU145NC cells. (c) Cell lysates from DU145 cells were immunoprecipitated using anti‐integrin α5 or β1 antibodies, and heterodimers were detected. I‐branching N‐acetylglucosaminyltransferase (GCNT2) expression had no effect on heterodimerization of α5β1 integrin. (d) Focal adhesion kinase (FAK) and AKT are downstream targets of integrin signaling. FAK phosphorylation at tyrosine 397 (p‐Y397) was similar in DU145NC and DU145GCNT2KD4 after integrin–fibronectin interactions. In contrast, AKT phosphorylation at serine 473 (p‐S473) was strongly inhibited by GCNT2 knockdown in DU145 cells. (e) A functional blocking antibody against α5 and β1 integrins inhibited α5β1 integrin–fibronectin interactions. Pretreatment of DU145NC and DU145GCNT2KD4 cells with anti‐integrin α5 or β1 led to lower FAK p‐Y397 and AKT p‐S473 levels compared with those after pretreatment with IgG isotype control. (f) Pretreatment of DU145NC cells with RGD peptide (100, 200, 400, or 800 μM) inhibited FAK p‐Y397 and AKT p‐S473 in a concentration‐dependent manner. IB, immunoblotting; IP, immunoprecipitation; NS, not significant; t‐FAK, total‐FAK; t‐AKT, total‐AKT.
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cas12859-fig-0004: I‐antigen regulates cell spread and α5β1 integrin–fibronectin interactions that are mediated by protein kinase B (AKT) phosphorylation in DU145NC and DU145GCNT2KD4 cells. Cell adhesion and spreading on fibronectin (FN) was examined by harvesting cells after a 30 min of culture on fibronectin‐coated 96‐well plates (a). DU145NC and DU145GCNT2KD4 cells showed no significant differences in adhesion potential. (b) Cells were harvested from fibronectin‐coated glass slides after 30 min of incubation, and spreading cells were visualized using crystal violet solution. Percentages of spreading cells were significantly lower in DU145GCNT2KD4 cells than in DU145NC cells. (c) Cell lysates from DU145 cells were immunoprecipitated using anti‐integrin α5 or β1 antibodies, and heterodimers were detected. I‐branching N‐acetylglucosaminyltransferase (GCNT2) expression had no effect on heterodimerization of α5β1 integrin. (d) Focal adhesion kinase (FAK) and AKT are downstream targets of integrin signaling. FAK phosphorylation at tyrosine 397 (p‐Y397) was similar in DU145NC and DU145GCNT2KD4 after integrin–fibronectin interactions. In contrast, AKT phosphorylation at serine 473 (p‐S473) was strongly inhibited by GCNT2 knockdown in DU145 cells. (e) A functional blocking antibody against α5 and β1 integrins inhibited α5β1 integrin–fibronectin interactions. Pretreatment of DU145NC and DU145GCNT2KD4 cells with anti‐integrin α5 or β1 led to lower FAK p‐Y397 and AKT p‐S473 levels compared with those after pretreatment with IgG isotype control. (f) Pretreatment of DU145NC cells with RGD peptide (100, 200, 400, or 800 μM) inhibited FAK p‐Y397 and AKT p‐S473 in a concentration‐dependent manner. IB, immunoblotting; IP, immunoprecipitation; NS, not significant; t‐FAK, total‐FAK; t‐AKT, total‐AKT.

Mentions: Integrin is a well‐known heterodimeric receptor of the ECM and has reported roles in cell adhesion.26 The major stromal ECM motility factor fibronectin has been shown to interact with the integrin α5β1 heterodimer in a glycan‐structure dependent manner.27, 28 In the present experiments, α5 integrin and β1 integrin were expressed at similar levels in DU145NC and GCNT2 knockdown cells (Fig. 3e). Thus, to confirm the role of I‐antigen in cell–ECM interactions, adhesion and spreading assays were carried out using DU145 cell lines on fibronectin‐coated plates. Although adhesion on fibronectin did not differ between DU145NC and DU145GCNT2KD4 cells, significantly fewer DU145GCNT2KD4 cells showed spreading activity (Fig. 4a,b). Integrin–ECM interactions that mediate outside–inside signals play important roles in cell spreading and migration.29, 30 Moreover, abnormal heterodimeric forms of these molecules have been shown to inhibit integrin‐mediated signaling.31, 32 However, heterodimeric forms of α5β1 integrin did not differ between GCNT2 knockdown and DU145NC cells (Fig. 4c). In previous studies, FAK and PI3K/AKT are reported downstream targets of integrin‐mediated signaling.29, 32, 33 Although FAK phosphorylation at tyrosine 397 (p‐Y397) was not affected by GCNT2 expression on fibronectin‐coated plates, AKT phosphorylation at serine 473 (p‐S473) was significantly less in DU145GCNT2KD4 cells than in DU145NC cells (Fig. 4d). Moreover, AKT p‐S473 was inhibited by antibody blocking and RGD peptide of the fibronectin–integrin interaction (Fig. 4e,f), suggesting that I‐antigens enhance integrin‐mediated PI3K/AKT signaling.


I-branching N-acetylglucosaminyltransferase regulates prostate cancer invasiveness by enhancing α5β1 integrin signaling.

Mikami J, Tobisawa Y, Yoneyama T, Hatakeyama S, Mori K, Hashimoto Y, Koie T, Ohyama C, Fukuda M - Cancer Sci. (2016)

I‐antigen regulates cell spread and α5β1 integrin–fibronectin interactions that are mediated by protein kinase B (AKT) phosphorylation in DU145NC and DU145GCNT2KD4 cells. Cell adhesion and spreading on fibronectin (FN) was examined by harvesting cells after a 30 min of culture on fibronectin‐coated 96‐well plates (a). DU145NC and DU145GCNT2KD4 cells showed no significant differences in adhesion potential. (b) Cells were harvested from fibronectin‐coated glass slides after 30 min of incubation, and spreading cells were visualized using crystal violet solution. Percentages of spreading cells were significantly lower in DU145GCNT2KD4 cells than in DU145NC cells. (c) Cell lysates from DU145 cells were immunoprecipitated using anti‐integrin α5 or β1 antibodies, and heterodimers were detected. I‐branching N‐acetylglucosaminyltransferase (GCNT2) expression had no effect on heterodimerization of α5β1 integrin. (d) Focal adhesion kinase (FAK) and AKT are downstream targets of integrin signaling. FAK phosphorylation at tyrosine 397 (p‐Y397) was similar in DU145NC and DU145GCNT2KD4 after integrin–fibronectin interactions. In contrast, AKT phosphorylation at serine 473 (p‐S473) was strongly inhibited by GCNT2 knockdown in DU145 cells. (e) A functional blocking antibody against α5 and β1 integrins inhibited α5β1 integrin–fibronectin interactions. Pretreatment of DU145NC and DU145GCNT2KD4 cells with anti‐integrin α5 or β1 led to lower FAK p‐Y397 and AKT p‐S473 levels compared with those after pretreatment with IgG isotype control. (f) Pretreatment of DU145NC cells with RGD peptide (100, 200, 400, or 800 μM) inhibited FAK p‐Y397 and AKT p‐S473 in a concentration‐dependent manner. IB, immunoblotting; IP, immunoprecipitation; NS, not significant; t‐FAK, total‐FAK; t‐AKT, total‐AKT.
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cas12859-fig-0004: I‐antigen regulates cell spread and α5β1 integrin–fibronectin interactions that are mediated by protein kinase B (AKT) phosphorylation in DU145NC and DU145GCNT2KD4 cells. Cell adhesion and spreading on fibronectin (FN) was examined by harvesting cells after a 30 min of culture on fibronectin‐coated 96‐well plates (a). DU145NC and DU145GCNT2KD4 cells showed no significant differences in adhesion potential. (b) Cells were harvested from fibronectin‐coated glass slides after 30 min of incubation, and spreading cells were visualized using crystal violet solution. Percentages of spreading cells were significantly lower in DU145GCNT2KD4 cells than in DU145NC cells. (c) Cell lysates from DU145 cells were immunoprecipitated using anti‐integrin α5 or β1 antibodies, and heterodimers were detected. I‐branching N‐acetylglucosaminyltransferase (GCNT2) expression had no effect on heterodimerization of α5β1 integrin. (d) Focal adhesion kinase (FAK) and AKT are downstream targets of integrin signaling. FAK phosphorylation at tyrosine 397 (p‐Y397) was similar in DU145NC and DU145GCNT2KD4 after integrin–fibronectin interactions. In contrast, AKT phosphorylation at serine 473 (p‐S473) was strongly inhibited by GCNT2 knockdown in DU145 cells. (e) A functional blocking antibody against α5 and β1 integrins inhibited α5β1 integrin–fibronectin interactions. Pretreatment of DU145NC and DU145GCNT2KD4 cells with anti‐integrin α5 or β1 led to lower FAK p‐Y397 and AKT p‐S473 levels compared with those after pretreatment with IgG isotype control. (f) Pretreatment of DU145NC cells with RGD peptide (100, 200, 400, or 800 μM) inhibited FAK p‐Y397 and AKT p‐S473 in a concentration‐dependent manner. IB, immunoblotting; IP, immunoprecipitation; NS, not significant; t‐FAK, total‐FAK; t‐AKT, total‐AKT.
Mentions: Integrin is a well‐known heterodimeric receptor of the ECM and has reported roles in cell adhesion.26 The major stromal ECM motility factor fibronectin has been shown to interact with the integrin α5β1 heterodimer in a glycan‐structure dependent manner.27, 28 In the present experiments, α5 integrin and β1 integrin were expressed at similar levels in DU145NC and GCNT2 knockdown cells (Fig. 3e). Thus, to confirm the role of I‐antigen in cell–ECM interactions, adhesion and spreading assays were carried out using DU145 cell lines on fibronectin‐coated plates. Although adhesion on fibronectin did not differ between DU145NC and DU145GCNT2KD4 cells, significantly fewer DU145GCNT2KD4 cells showed spreading activity (Fig. 4a,b). Integrin–ECM interactions that mediate outside–inside signals play important roles in cell spreading and migration.29, 30 Moreover, abnormal heterodimeric forms of these molecules have been shown to inhibit integrin‐mediated signaling.31, 32 However, heterodimeric forms of α5β1 integrin did not differ between GCNT2 knockdown and DU145NC cells (Fig. 4c). In previous studies, FAK and PI3K/AKT are reported downstream targets of integrin‐mediated signaling.29, 32, 33 Although FAK phosphorylation at tyrosine 397 (p‐Y397) was not affected by GCNT2 expression on fibronectin‐coated plates, AKT phosphorylation at serine 473 (p‐S473) was significantly less in DU145GCNT2KD4 cells than in DU145NC cells (Fig. 4d). Moreover, AKT p‐S473 was inhibited by antibody blocking and RGD peptide of the fibronectin–integrin interaction (Fig. 4e,f), suggesting that I‐antigens enhance integrin‐mediated PI3K/AKT signaling.

Bottom Line: GCNT2-positive cells were significantly lesser in organ-confined disease than in that with extra-capsular extensions, and GCNT2-negative tumors were associated with significantly better prostate-specific antigen-free survival compared with GCNT2-positive tumors.Subsequent functional studies revealed that knockdown of GCNT2 expression in PCa cell lines significantly inhibited cell migration and invasion.In conclusion, GCNT2 expression is closely associated with invasive potential of PCa.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.

Show MeSH
Related in: MedlinePlus