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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 controls prostate cancer cell invasion. (a) Messenger RNA expression of GCNT2 was determined in DU145 cells using quantitative PCR. GCNT2 expression levels were normalized to those of human G3PDH. Clone 3 and Clone 4 (DU145GCNT2KD3 and DU145GCNT2KD4) showed >70% inhibition of GCNT2 expression. (b) Protein expression of GCNT2 was determined in DU145‐derived cells using Western blotting. The GCNT2 expression level was lower in DU145GCNT2KD3 and 4 than in DU145NC. (c) In vitro cell proliferation was similar in DU145GCNT2KD3 and 4 cells at day 7, and strongly reduced cell migration (d) and invasion (e) was observed in Transwell assays. Assays were carried out in triplicate. *P < 0.05.
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cas12859-fig-0002: I‐branching N‐acetylglucosaminyltransferase (GCNT2) expression controls prostate cancer cell invasion. (a) Messenger RNA expression of GCNT2 was determined in DU145 cells using quantitative PCR. GCNT2 expression levels were normalized to those of human G3PDH. Clone 3 and Clone 4 (DU145GCNT2KD3 and DU145GCNT2KD4) showed >70% inhibition of GCNT2 expression. (b) Protein expression of GCNT2 was determined in DU145‐derived cells using Western blotting. The GCNT2 expression level was lower in DU145GCNT2KD3 and 4 than in DU145NC. (c) In vitro cell proliferation was similar in DU145GCNT2KD3 and 4 cells at day 7, and strongly reduced cell migration (d) and invasion (e) was observed in Transwell assays. Assays were carried out in triplicate. *P < 0.05.

Mentions: To investigate the role of GCNT2 expression in PCa cells, we established GCNT2 knockdown DU145 cell lines. In subsequent qPCR analyses and Western blotting, clone 3 and clone 4 showed >70% inhibition of GCNT2 expression (Fig. 2a,b). Although GCNT2 knockdown significantly inhibited cell proliferation at day 3 in clone 3 (DU145GCNT2KD3), total cell numbers at day 7 did not differ between siControl (DU145NC), DU145GCNT2KD3, and clone 4 (DU145GCNT2KD4) cells (Fig. 2c). These results suggest that GCNT2 expression is not critical for cell proliferation in vitro. In subsequent experiments, the effects of GCNT2 expression were examined using migration and invasion assays in DU145NC, GCNT2KD3, and GCNT2KD4 cells using a Transwell system. In comparisons with DU145NC cells, migration and invasion was strongly inhibited in GCNT2 knockdown cell lines (Fig. 2d,e). In further experiments, GCNT2 expression was transiently inhibited using siRNA transfection in PC3 cells and resulted in decreased invasion potential (Fig. S2a). Moreover, wound healing assays showed significantly decreased surface coverage rates in GCNT2 knockdown cell lines compared with that in DU145NC cells (Fig. S2b). In a previous study, high expression of GCNT2 was associated with EMT and accelerated cell invasion in breast cancers.19 In agreement, comparisons of the present DU145NC and DU145GCNT2KD4 cell lines with PC3 cells revealed similar patterns of EMT marker expression (Fig. S2c), suggesting that GCNT2 regulates migration and invasion without stimulating EMT in PCa cells.


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 controls prostate cancer cell invasion. (a) Messenger RNA expression of GCNT2 was determined in DU145 cells using quantitative PCR. GCNT2 expression levels were normalized to those of human G3PDH. Clone 3 and Clone 4 (DU145GCNT2KD3 and DU145GCNT2KD4) showed >70% inhibition of GCNT2 expression. (b) Protein expression of GCNT2 was determined in DU145‐derived cells using Western blotting. The GCNT2 expression level was lower in DU145GCNT2KD3 and 4 than in DU145NC. (c) In vitro cell proliferation was similar in DU145GCNT2KD3 and 4 cells at day 7, and strongly reduced cell migration (d) and invasion (e) was observed in Transwell assays. Assays were carried out in triplicate. *P < 0.05.
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Related In: Results  -  Collection

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cas12859-fig-0002: I‐branching N‐acetylglucosaminyltransferase (GCNT2) expression controls prostate cancer cell invasion. (a) Messenger RNA expression of GCNT2 was determined in DU145 cells using quantitative PCR. GCNT2 expression levels were normalized to those of human G3PDH. Clone 3 and Clone 4 (DU145GCNT2KD3 and DU145GCNT2KD4) showed >70% inhibition of GCNT2 expression. (b) Protein expression of GCNT2 was determined in DU145‐derived cells using Western blotting. The GCNT2 expression level was lower in DU145GCNT2KD3 and 4 than in DU145NC. (c) In vitro cell proliferation was similar in DU145GCNT2KD3 and 4 cells at day 7, and strongly reduced cell migration (d) and invasion (e) was observed in Transwell assays. Assays were carried out in triplicate. *P < 0.05.
Mentions: To investigate the role of GCNT2 expression in PCa cells, we established GCNT2 knockdown DU145 cell lines. In subsequent qPCR analyses and Western blotting, clone 3 and clone 4 showed >70% inhibition of GCNT2 expression (Fig. 2a,b). Although GCNT2 knockdown significantly inhibited cell proliferation at day 3 in clone 3 (DU145GCNT2KD3), total cell numbers at day 7 did not differ between siControl (DU145NC), DU145GCNT2KD3, and clone 4 (DU145GCNT2KD4) cells (Fig. 2c). These results suggest that GCNT2 expression is not critical for cell proliferation in vitro. In subsequent experiments, the effects of GCNT2 expression were examined using migration and invasion assays in DU145NC, GCNT2KD3, and GCNT2KD4 cells using a Transwell system. In comparisons with DU145NC cells, migration and invasion was strongly inhibited in GCNT2 knockdown cell lines (Fig. 2d,e). In further experiments, GCNT2 expression was transiently inhibited using siRNA transfection in PC3 cells and resulted in decreased invasion potential (Fig. S2a). Moreover, wound healing assays showed significantly decreased surface coverage rates in GCNT2 knockdown cell lines compared with that in DU145NC cells (Fig. S2b). In a previous study, high expression of GCNT2 was associated with EMT and accelerated cell invasion in breast cancers.19 In agreement, comparisons of the present DU145NC and DU145GCNT2KD4 cell lines with PC3 cells revealed similar patterns of EMT marker expression (Fig. S2c), suggesting that GCNT2 regulates migration and invasion without stimulating EMT in PCa cells.

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