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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‐branching N‐acetylglucosaminyltransferase (GCNT2) expression regulates I‐antigen presentation on prostate cancer cells. Cell surface I‐antigen expression was determined using flow cytometry (FC) and immunocytochemistry with anti‐I antigen human antisera (AS). (a) DU145GCNT2KD3 and DU145GCNT2KD4 showed decreased cell surface I‐antigen expression in FC analyses. (b) DU145NC and DU145GCNT2KD4 cells were cultured on glass slides and were stained with anti‐I antigen human antisera or the human IgM isotype control. Brown indicates I‐antigen expression; blue indicates nuclear staining. DU145GCNT2KD4 cells had strongly reduced I‐antigen expression. DU145 cells were cultured with benzyl‐α‐GalNAc (BAG), tunycamycin (TM), or DL‐threo‐1‐Phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanol hydrochloride (PPMP) for 48 h. I‐antigen expression was determined using immunocytochemistry (c) and FC (d). I‐antigen expression was significantly reduced in BAG‐treated cells and PPMP‐treated cells. TM‐treated cells had no effect on I‐antigen presentation. Co‐treatment with BAG and PPMP strongly reduced I‐antigen expression compared with either treatment alone. Population comparison was carried out using Flowjo software. Assays were carried out in triplicate. (e) Integrin expression was determined using FC, and expression of α5 and β1 integrins was similar in DU145NC and GCNT2 knockdown cell lines. NS, not significant; pAb, polyclonal antibody. Scale bar = 200 μm.
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cas12859-fig-0003: I‐branching N‐acetylglucosaminyltransferase (GCNT2) expression regulates I‐antigen presentation on prostate cancer cells. Cell surface I‐antigen expression was determined using flow cytometry (FC) and immunocytochemistry with anti‐I antigen human antisera (AS). (a) DU145GCNT2KD3 and DU145GCNT2KD4 showed decreased cell surface I‐antigen expression in FC analyses. (b) DU145NC and DU145GCNT2KD4 cells were cultured on glass slides and were stained with anti‐I antigen human antisera or the human IgM isotype control. Brown indicates I‐antigen expression; blue indicates nuclear staining. DU145GCNT2KD4 cells had strongly reduced I‐antigen expression. DU145 cells were cultured with benzyl‐α‐GalNAc (BAG), tunycamycin (TM), or DL‐threo‐1‐Phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanol hydrochloride (PPMP) for 48 h. I‐antigen expression was determined using immunocytochemistry (c) and FC (d). I‐antigen expression was significantly reduced in BAG‐treated cells and PPMP‐treated cells. TM‐treated cells had no effect on I‐antigen presentation. Co‐treatment with BAG and PPMP strongly reduced I‐antigen expression compared with either treatment alone. Population comparison was carried out using Flowjo software. Assays were carried out in triplicate. (e) Integrin expression was determined using FC, and expression of α5 and β1 integrins was similar in DU145NC and GCNT2 knockdown cell lines. NS, not significant; pAb, polyclonal antibody. Scale bar = 200 μm.

Mentions: Interactions between cells and the ECM have been shown to regulate cell motility.23 Moreover, cell surface glycan modifications have reported biological functions during adhesion to the ECM and selectins and inhibits natural killer cell cytotoxicity.24, 25 Thus, we investigated the effects of GCNT2 on cell surface glycans, and confirmed that GCNT2 converts i‐antigen to I‐antigen on cell surface carbohydrate structures (Fig. 1a). Specifically, GCNT2‐expressing PCa cell lines showed pronounced cell surface presentation of I‐antigen (Fig. S3a,b). In contrast, GCNT2 knockdown cells had limited I‐antigen presentation on cell surfaces (Fig. 3a,b). GCNT2 reportedly acts on O‐glycans, N‐glycans, and glycolipids to form GlcNAc‐Gal branches (Fig. 1a).12 Accordingly, after treatment of DU145NC cells with the inhibitors BAG or PPMP, significantly decreased I‐antigen expression was observed. Moreover, co‐treatment with BAG and PPMP led to greater inhibition of I‐antigen presentation than individual treatments (Fig. 3d). In contrast, treatment with TM did not decrease I‐antigen presentation on cell surfaces (Fig. 3c,d). Treatment with these inhibitors decreased each glycan on the cell surface (Fig. S4), suggesting that I‐antigens were formed on O‐glycan and glycolipid molecules on PCa cell surfaces.


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‐branching N‐acetylglucosaminyltransferase (GCNT2) expression regulates I‐antigen presentation on prostate cancer cells. Cell surface I‐antigen expression was determined using flow cytometry (FC) and immunocytochemistry with anti‐I antigen human antisera (AS). (a) DU145GCNT2KD3 and DU145GCNT2KD4 showed decreased cell surface I‐antigen expression in FC analyses. (b) DU145NC and DU145GCNT2KD4 cells were cultured on glass slides and were stained with anti‐I antigen human antisera or the human IgM isotype control. Brown indicates I‐antigen expression; blue indicates nuclear staining. DU145GCNT2KD4 cells had strongly reduced I‐antigen expression. DU145 cells were cultured with benzyl‐α‐GalNAc (BAG), tunycamycin (TM), or DL‐threo‐1‐Phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanol hydrochloride (PPMP) for 48 h. I‐antigen expression was determined using immunocytochemistry (c) and FC (d). I‐antigen expression was significantly reduced in BAG‐treated cells and PPMP‐treated cells. TM‐treated cells had no effect on I‐antigen presentation. Co‐treatment with BAG and PPMP strongly reduced I‐antigen expression compared with either treatment alone. Population comparison was carried out using Flowjo software. Assays were carried out in triplicate. (e) Integrin expression was determined using FC, and expression of α5 and β1 integrins was similar in DU145NC and GCNT2 knockdown cell lines. NS, not significant; pAb, polyclonal antibody. Scale bar = 200 μm.
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cas12859-fig-0003: I‐branching N‐acetylglucosaminyltransferase (GCNT2) expression regulates I‐antigen presentation on prostate cancer cells. Cell surface I‐antigen expression was determined using flow cytometry (FC) and immunocytochemistry with anti‐I antigen human antisera (AS). (a) DU145GCNT2KD3 and DU145GCNT2KD4 showed decreased cell surface I‐antigen expression in FC analyses. (b) DU145NC and DU145GCNT2KD4 cells were cultured on glass slides and were stained with anti‐I antigen human antisera or the human IgM isotype control. Brown indicates I‐antigen expression; blue indicates nuclear staining. DU145GCNT2KD4 cells had strongly reduced I‐antigen expression. DU145 cells were cultured with benzyl‐α‐GalNAc (BAG), tunycamycin (TM), or DL‐threo‐1‐Phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanol hydrochloride (PPMP) for 48 h. I‐antigen expression was determined using immunocytochemistry (c) and FC (d). I‐antigen expression was significantly reduced in BAG‐treated cells and PPMP‐treated cells. TM‐treated cells had no effect on I‐antigen presentation. Co‐treatment with BAG and PPMP strongly reduced I‐antigen expression compared with either treatment alone. Population comparison was carried out using Flowjo software. Assays were carried out in triplicate. (e) Integrin expression was determined using FC, and expression of α5 and β1 integrins was similar in DU145NC and GCNT2 knockdown cell lines. NS, not significant; pAb, polyclonal antibody. Scale bar = 200 μm.
Mentions: Interactions between cells and the ECM have been shown to regulate cell motility.23 Moreover, cell surface glycan modifications have reported biological functions during adhesion to the ECM and selectins and inhibits natural killer cell cytotoxicity.24, 25 Thus, we investigated the effects of GCNT2 on cell surface glycans, and confirmed that GCNT2 converts i‐antigen to I‐antigen on cell surface carbohydrate structures (Fig. 1a). Specifically, GCNT2‐expressing PCa cell lines showed pronounced cell surface presentation of I‐antigen (Fig. S3a,b). In contrast, GCNT2 knockdown cells had limited I‐antigen presentation on cell surfaces (Fig. 3a,b). GCNT2 reportedly acts on O‐glycans, N‐glycans, and glycolipids to form GlcNAc‐Gal branches (Fig. 1a).12 Accordingly, after treatment of DU145NC cells with the inhibitors BAG or PPMP, significantly decreased I‐antigen expression was observed. Moreover, co‐treatment with BAG and PPMP led to greater inhibition of I‐antigen presentation than individual treatments (Fig. 3d). In contrast, treatment with TM did not decrease I‐antigen presentation on cell surfaces (Fig. 3c,d). Treatment with these inhibitors decreased each glycan on the cell surface (Fig. S4), suggesting that I‐antigens were formed on O‐glycan and glycolipid molecules on PCa cell surfaces.

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