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COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer.

Hofmann BT, Schlüter L, Lange P, Mercanoglu B, Ewald F, Fölster A, Picksak AS, Harder S, El Gammal AT, Grupp K, Güngör C, Drenckhan A, Schlüter H, Wagener C, Izbicki JR, Jücker M, Bockhorn M, Wolters-Eisfeld G - Mol. Cancer (2015)

Bottom Line: Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations.Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells.Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany. bi.hofmann@uke.de.

ABSTRACT

Background: Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties.

Methods: Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data.

Results: Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037).

Conclusion: This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.

No MeSH data available.


Related in: MedlinePlus

Aberrant mRNA expression levels of GalNAc-transferases, T-Synthase and COSMC as well as T-synthase activity in COSMC-depleted pancreatic cancer cells. a Relative mRNA expression of all human GalNAc-transferase isoforms in COSMC knockdown as well as control Panc-1 cells, quantified using real-time PCR. b Relative quantification of T-synthase mRNA expression in COSMC knockdown and control Panc-1 cells. Note: mRNA expression level of T-synthase is almost doubled in COSMC knockdown cells. c Relative mRNA expression of GalNAc-transferase isoforms in L3.6pl. d Relative quantification of T-synthase and COSMC mRNA expression in L3.6pl COSMC knockdown and control cells. e T-synthase activity was measured in Panc-1 and L3.6pl COSMC knockdown and control cells using GalNAc-α-4MU fluorescent assay
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Fig2: Aberrant mRNA expression levels of GalNAc-transferases, T-Synthase and COSMC as well as T-synthase activity in COSMC-depleted pancreatic cancer cells. a Relative mRNA expression of all human GalNAc-transferase isoforms in COSMC knockdown as well as control Panc-1 cells, quantified using real-time PCR. b Relative quantification of T-synthase mRNA expression in COSMC knockdown and control Panc-1 cells. Note: mRNA expression level of T-synthase is almost doubled in COSMC knockdown cells. c Relative mRNA expression of GalNAc-transferase isoforms in L3.6pl. d Relative quantification of T-synthase and COSMC mRNA expression in L3.6pl COSMC knockdown and control cells. e T-synthase activity was measured in Panc-1 and L3.6pl COSMC knockdown and control cells using GalNAc-α-4MU fluorescent assay

Mentions: In order to induce the expression of Tn antigen in the minimal Tn antigen positive Panc-1 cell line and the moderate Tn antigen positive L3.6pl cell line, we performed a lentiviral-mediated knockdown of COSMC chaperone followed by single colony selection with puromycin. Knockdown of COSMC was highly efficient in Panc-1 cells, compared to cells transduced with a control vector (Additional file 1: Figure S4). Additionally, down regulation of COSMC was confirmed on mRNA level by real-time PCR, achieving 97 % efficiency in Panc-1 and 73 % in L3.6pl cells (Fig. 2b and d). Next, expression of atypical O-glycans was evaluated using different glyco epitope recognizing antibodies and lectins. Using sialyl-Tn and Tn antigen antibodies as well as VVL and WFL, revealed an enhancement of distinct staining patterns in COSMC knockdown cells compared to control cells (Fig. 1c). L3.6pl cells revealed an enhanced Tn antigen expression only after treatment with Neuraminidase (Additional file 1: Figure S5). Immunocytochemistry revealed an enhanced staining of VVL and WFL lectin in COSMC knockdown cells compared to controls (Additional file 1: Figure S6).Fig. 2


COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer.

Hofmann BT, Schlüter L, Lange P, Mercanoglu B, Ewald F, Fölster A, Picksak AS, Harder S, El Gammal AT, Grupp K, Güngör C, Drenckhan A, Schlüter H, Wagener C, Izbicki JR, Jücker M, Bockhorn M, Wolters-Eisfeld G - Mol. Cancer (2015)

Aberrant mRNA expression levels of GalNAc-transferases, T-Synthase and COSMC as well as T-synthase activity in COSMC-depleted pancreatic cancer cells. a Relative mRNA expression of all human GalNAc-transferase isoforms in COSMC knockdown as well as control Panc-1 cells, quantified using real-time PCR. b Relative quantification of T-synthase mRNA expression in COSMC knockdown and control Panc-1 cells. Note: mRNA expression level of T-synthase is almost doubled in COSMC knockdown cells. c Relative mRNA expression of GalNAc-transferase isoforms in L3.6pl. d Relative quantification of T-synthase and COSMC mRNA expression in L3.6pl COSMC knockdown and control cells. e T-synthase activity was measured in Panc-1 and L3.6pl COSMC knockdown and control cells using GalNAc-α-4MU fluorescent assay
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4447007&req=5

Fig2: Aberrant mRNA expression levels of GalNAc-transferases, T-Synthase and COSMC as well as T-synthase activity in COSMC-depleted pancreatic cancer cells. a Relative mRNA expression of all human GalNAc-transferase isoforms in COSMC knockdown as well as control Panc-1 cells, quantified using real-time PCR. b Relative quantification of T-synthase mRNA expression in COSMC knockdown and control Panc-1 cells. Note: mRNA expression level of T-synthase is almost doubled in COSMC knockdown cells. c Relative mRNA expression of GalNAc-transferase isoforms in L3.6pl. d Relative quantification of T-synthase and COSMC mRNA expression in L3.6pl COSMC knockdown and control cells. e T-synthase activity was measured in Panc-1 and L3.6pl COSMC knockdown and control cells using GalNAc-α-4MU fluorescent assay
Mentions: In order to induce the expression of Tn antigen in the minimal Tn antigen positive Panc-1 cell line and the moderate Tn antigen positive L3.6pl cell line, we performed a lentiviral-mediated knockdown of COSMC chaperone followed by single colony selection with puromycin. Knockdown of COSMC was highly efficient in Panc-1 cells, compared to cells transduced with a control vector (Additional file 1: Figure S4). Additionally, down regulation of COSMC was confirmed on mRNA level by real-time PCR, achieving 97 % efficiency in Panc-1 and 73 % in L3.6pl cells (Fig. 2b and d). Next, expression of atypical O-glycans was evaluated using different glyco epitope recognizing antibodies and lectins. Using sialyl-Tn and Tn antigen antibodies as well as VVL and WFL, revealed an enhancement of distinct staining patterns in COSMC knockdown cells compared to control cells (Fig. 1c). L3.6pl cells revealed an enhanced Tn antigen expression only after treatment with Neuraminidase (Additional file 1: Figure S5). Immunocytochemistry revealed an enhanced staining of VVL and WFL lectin in COSMC knockdown cells compared to controls (Additional file 1: Figure S6).Fig. 2

Bottom Line: Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations.Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells.Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines.

View Article: PubMed Central - PubMed

Affiliation: Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany. bi.hofmann@uke.de.

ABSTRACT

Background: Human pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies in the world and despite great efforts in research types of treatment remain limited. A frequently detected alteration in PDACs is a truncated O-linked N-acetylgalactosamine (GalNAc) glycosylation with expression of the Tn antigen. Changes in O-glycosylation affect posttranslationally modified O-GalNAc proteins resulting in profound cellular alterations. Tn antigen is a tumor associated glycan detected in 75-90 % of PDACs and up to 67 % in its precursor lesions. Since the role of Tn antigen expression in PDAC is insufficiently understood we analyzed the impact of COSMC mediated Tn antigen expression in two human PDAC cell lines on cellular oncogenic properties.

Methods: Forced expression of Tn antigen on O-glycosylated proteins in pancreatic cancer cells was induced by lentiviral-mediated knockdown of the COSMC chaperone, which prevented O-glycan elongation beyond the initial GalNAcα1- residue on O-linked glycoproteins. Altered O-GalNAc glycosylation was analyzed in human pancreatic cancer cell lines Panc-1 and L3.6pl using Western and Far-Western blot as well as immunocytochemical techniques. To assess the biological implications of COSMC function on oncogenic properties, cell viability assays, scratch assays combined with live cell imaging, migration and apoptosis assays were performed. Lectin based glycoprotein enrichment with subsequent mass spectrometric analysis identified new cancer O-GalNAc modified proteins. Expression of Tn antigen bearing Nucleolin in patient derived PDAC tumor specimens was evaluated and correlated with clinicopathological data.

Results: Tn antigen expression was induced on various O-GalNAc glycoproteins in COSMC deficient cell lines compared to the control. Proliferation was reduced (p < 0.001) in COSMC knockdown cells, whereas migration was increased (p < 0.001) and apoptosis was decreased (p = 0.03), highlighting the importance of Tn antigen expression on metastatic and anti-apoptotic behavior of PDAC derived cells. Nucleolin was identified as O-GalNAc modified protein in COSMC deficient PDAC cell lines. Interestingly, immunohistochemical staining and co-localization studies of patient derived PDACs revealed poor survival for patients with strong co-localization of Tn antigen and Nucleolin (p = 0.037).

Conclusion: This study substantiates the influence of altered O-glycan (Tn/STn) expression on oncogenic properties in pancreatic cancer and identifies O-GalNAc modified Nucleolin as novel prognostic marker.

No MeSH data available.


Related in: MedlinePlus