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C1GALT1 promotes invasive phenotypes of hepatocellular carcinoma cells by modulating integrin β1 glycosylation and activity.

Liu CH, Hu RH, Huang MJ, Lai IR, Chen CH, Lai HS, Wu YM, Huang MC - PLoS ONE (2014)

Bottom Line: In this study, we found that overexpression of C1GALT1 enhanced HCC cell adhesion to extracellular matrix (ECM) proteins, migration, and invasion, whereas RNAi-mediated knockdown of C1GALT1 suppressed these phenotypes.Mechanistic investigations showed that the C1GALT1-enhanced phenotypic changes in HCC cells were significantly suppressed by anti-integrin β1 blocking antibody.These results suggest that C1GALT1 could enhance HCC invasiveness through integrin β1 and provide novel insights into the roles of O-glycosylation in HCC metastasis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Anatomy and Cell Biology, National Taiwan University College of Medicine, Taipei, Taiwan.

ABSTRACT
Cancer cell invasion and metastasis are the primary causes of treatment failure and death in hepatocellular carcinoma (HCC). We previously reported that core 1 β1,3-galactosyltransferase (C1GALT1) is frequently overexpressed in HCC tumors and its expression is associated with advanced tumor stage, metastasis, and poor survival. However, the underlying mechanisms of C1GALT1 in HCC malignancy remain unclear. In this study, we found that overexpression of C1GALT1 enhanced HCC cell adhesion to extracellular matrix (ECM) proteins, migration, and invasion, whereas RNAi-mediated knockdown of C1GALT1 suppressed these phenotypes. The promoting effect of C1GALT1 on the metastasis of HCC cells was demonstrated in a mouse xenograft model. Mechanistic investigations showed that the C1GALT1-enhanced phenotypic changes in HCC cells were significantly suppressed by anti-integrin β1 blocking antibody. Moreover, C1GALT1 was able to modify O-glycans on integrin β1 and regulate integrin β1 activity as well as its downstream signaling. These results suggest that C1GALT1 could enhance HCC invasiveness through integrin β1 and provide novel insights into the roles of O-glycosylation in HCC metastasis.

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C1GALT1 regulates lung metastasis of HCC cells in NOD/SCID mice.(a) Stable overexpression and knockdown of C1GALT1 in HCC cells. The protein expression levels of C1GALT1 were analyzed by Western blotting. (b) Effects of C1GALT1 on lung metastasis. Metastatic tumors were increased in the C1GALT1 overexpressed group (left) and decreased in the C1GALT1 knockdown groups (right). Representative image of the excised lungs are shown at the bottom, n = 6 for each group. Results are shown as means ± SD. * P<0.05; **P<0.01. Blue arrows indicate the location of the tumor nodules on the lung surface. (c) H&E staining and immunohistochemistry of paraffin-embedded lung sections. Representative images (upper) and amplified images (middle) are shown. Immunostaining revealed the expression of C1GALT1 in metastatic tumors (bottom). The red dash line indicates the location of metastatic tumors. Scale bars  = 200 µm.
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pone-0094995-g002: C1GALT1 regulates lung metastasis of HCC cells in NOD/SCID mice.(a) Stable overexpression and knockdown of C1GALT1 in HCC cells. The protein expression levels of C1GALT1 were analyzed by Western blotting. (b) Effects of C1GALT1 on lung metastasis. Metastatic tumors were increased in the C1GALT1 overexpressed group (left) and decreased in the C1GALT1 knockdown groups (right). Representative image of the excised lungs are shown at the bottom, n = 6 for each group. Results are shown as means ± SD. * P<0.05; **P<0.01. Blue arrows indicate the location of the tumor nodules on the lung surface. (c) H&E staining and immunohistochemistry of paraffin-embedded lung sections. Representative images (upper) and amplified images (middle) are shown. Immunostaining revealed the expression of C1GALT1 in metastatic tumors (bottom). The red dash line indicates the location of metastatic tumors. Scale bars  = 200 µm.

Mentions: To analyze the effects of C1GALT1 on HCC cell metastasis in vivo, a mouse tail vein injection model was used. C1GALT1 was stably overexpressed in HCC36 cells and knocked down in HA22T cells (Fig. 2a). To measure the carbohydrate changes on the stable transfectants, flow cytometry with FITC-conjugated PNA was performed [22], [23]. Overexpression of C1GALT1 increased PNA binding to the surface of HCC cells, whereas knockdown of C1GALT1 decreased PNA binding (Figure S1), indicating that C1GALT1 catalyzes the formation of T antigen on the cell surface. The stable clones were injected into NOD/SCID mice, which were sacrificed after 8 weeks. The results showed that overexpression of C1GALT1 significantly enhanced the number of lung metastatic tumors, while knockdown of C1GALT1 reduced the number of lung metastatic nodules (Fig. 2b). H&E staining confirmed the location of tumors in the lung sections, and immunohistochemical results confirmed the overexpression or knockdown of C1GALT1 in HCC metastatic tumor nodules (Fig. 2c). These results suggest that C1GALT1 enhances the metastatic potential of HCC cells in vivo.


C1GALT1 promotes invasive phenotypes of hepatocellular carcinoma cells by modulating integrin β1 glycosylation and activity.

Liu CH, Hu RH, Huang MJ, Lai IR, Chen CH, Lai HS, Wu YM, Huang MC - PLoS ONE (2014)

C1GALT1 regulates lung metastasis of HCC cells in NOD/SCID mice.(a) Stable overexpression and knockdown of C1GALT1 in HCC cells. The protein expression levels of C1GALT1 were analyzed by Western blotting. (b) Effects of C1GALT1 on lung metastasis. Metastatic tumors were increased in the C1GALT1 overexpressed group (left) and decreased in the C1GALT1 knockdown groups (right). Representative image of the excised lungs are shown at the bottom, n = 6 for each group. Results are shown as means ± SD. * P<0.05; **P<0.01. Blue arrows indicate the location of the tumor nodules on the lung surface. (c) H&E staining and immunohistochemistry of paraffin-embedded lung sections. Representative images (upper) and amplified images (middle) are shown. Immunostaining revealed the expression of C1GALT1 in metastatic tumors (bottom). The red dash line indicates the location of metastatic tumors. Scale bars  = 200 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4121071&req=5

pone-0094995-g002: C1GALT1 regulates lung metastasis of HCC cells in NOD/SCID mice.(a) Stable overexpression and knockdown of C1GALT1 in HCC cells. The protein expression levels of C1GALT1 were analyzed by Western blotting. (b) Effects of C1GALT1 on lung metastasis. Metastatic tumors were increased in the C1GALT1 overexpressed group (left) and decreased in the C1GALT1 knockdown groups (right). Representative image of the excised lungs are shown at the bottom, n = 6 for each group. Results are shown as means ± SD. * P<0.05; **P<0.01. Blue arrows indicate the location of the tumor nodules on the lung surface. (c) H&E staining and immunohistochemistry of paraffin-embedded lung sections. Representative images (upper) and amplified images (middle) are shown. Immunostaining revealed the expression of C1GALT1 in metastatic tumors (bottom). The red dash line indicates the location of metastatic tumors. Scale bars  = 200 µm.
Mentions: To analyze the effects of C1GALT1 on HCC cell metastasis in vivo, a mouse tail vein injection model was used. C1GALT1 was stably overexpressed in HCC36 cells and knocked down in HA22T cells (Fig. 2a). To measure the carbohydrate changes on the stable transfectants, flow cytometry with FITC-conjugated PNA was performed [22], [23]. Overexpression of C1GALT1 increased PNA binding to the surface of HCC cells, whereas knockdown of C1GALT1 decreased PNA binding (Figure S1), indicating that C1GALT1 catalyzes the formation of T antigen on the cell surface. The stable clones were injected into NOD/SCID mice, which were sacrificed after 8 weeks. The results showed that overexpression of C1GALT1 significantly enhanced the number of lung metastatic tumors, while knockdown of C1GALT1 reduced the number of lung metastatic nodules (Fig. 2b). H&E staining confirmed the location of tumors in the lung sections, and immunohistochemical results confirmed the overexpression or knockdown of C1GALT1 in HCC metastatic tumor nodules (Fig. 2c). These results suggest that C1GALT1 enhances the metastatic potential of HCC cells in vivo.

Bottom Line: In this study, we found that overexpression of C1GALT1 enhanced HCC cell adhesion to extracellular matrix (ECM) proteins, migration, and invasion, whereas RNAi-mediated knockdown of C1GALT1 suppressed these phenotypes.Mechanistic investigations showed that the C1GALT1-enhanced phenotypic changes in HCC cells were significantly suppressed by anti-integrin β1 blocking antibody.These results suggest that C1GALT1 could enhance HCC invasiveness through integrin β1 and provide novel insights into the roles of O-glycosylation in HCC metastasis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Anatomy and Cell Biology, National Taiwan University College of Medicine, Taipei, Taiwan.

ABSTRACT
Cancer cell invasion and metastasis are the primary causes of treatment failure and death in hepatocellular carcinoma (HCC). We previously reported that core 1 β1,3-galactosyltransferase (C1GALT1) is frequently overexpressed in HCC tumors and its expression is associated with advanced tumor stage, metastasis, and poor survival. However, the underlying mechanisms of C1GALT1 in HCC malignancy remain unclear. In this study, we found that overexpression of C1GALT1 enhanced HCC cell adhesion to extracellular matrix (ECM) proteins, migration, and invasion, whereas RNAi-mediated knockdown of C1GALT1 suppressed these phenotypes. The promoting effect of C1GALT1 on the metastasis of HCC cells was demonstrated in a mouse xenograft model. Mechanistic investigations showed that the C1GALT1-enhanced phenotypic changes in HCC cells were significantly suppressed by anti-integrin β1 blocking antibody. Moreover, C1GALT1 was able to modify O-glycans on integrin β1 and regulate integrin β1 activity as well as its downstream signaling. These results suggest that C1GALT1 could enhance HCC invasiveness through integrin β1 and provide novel insights into the roles of O-glycosylation in HCC metastasis.

Show MeSH
Related in: MedlinePlus