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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

Expression of aberrant O-glycans in pancreatic cancer. a Biosynthesis of Tn antigen, sTn antigen and Core1 and 3 structures. Tn antigen is composed of an O-glycosidic linked N-acetylgalactosamine (GalNAc) to the –OH group of serine/threonine (S/T). Tn antigen is either processed by core 1 T-synthase (C1GalT1) and its chaperone (COSMC), which transfers a galactose (Gal) to GalNAc-serine/threonine to form the T antigen also referred as core 1 structure or processed by transfer of a N-acetylglucosamine (GlcNAc) to form the core 3 structure. Tn antigen can also be modified by addition of a sialic acid (NeuAc). b Differential expression of Tn antigen in pancreatic carcinoma cell lines. Eight different PDAC cell lines were available for analysis. Western and Far-Western blot analysis of total cell lysates was performed using the Tn antigen specific antibody MA1-80055. Detection of HSC70 served as loading control. Jurkat cells were used as positive control for Tn antigen expression. c Expression of Tn antigen and aberrant O-glycans in COSMC knockdown cells. Western blot analysis showed a strong expression of aberrant O-glycans as well as Tn antigen in Panc-1 COSMC knockdown cells compared to control cells. Sialyl-Tn and Tn antibodies were used as well as lectins such as VVL (Vicia villosa lectin) and WFL (Wisteria floribunda lectin)
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Fig1: Expression of aberrant O-glycans in pancreatic cancer. a Biosynthesis of Tn antigen, sTn antigen and Core1 and 3 structures. Tn antigen is composed of an O-glycosidic linked N-acetylgalactosamine (GalNAc) to the –OH group of serine/threonine (S/T). Tn antigen is either processed by core 1 T-synthase (C1GalT1) and its chaperone (COSMC), which transfers a galactose (Gal) to GalNAc-serine/threonine to form the T antigen also referred as core 1 structure or processed by transfer of a N-acetylglucosamine (GlcNAc) to form the core 3 structure. Tn antigen can also be modified by addition of a sialic acid (NeuAc). b Differential expression of Tn antigen in pancreatic carcinoma cell lines. Eight different PDAC cell lines were available for analysis. Western and Far-Western blot analysis of total cell lysates was performed using the Tn antigen specific antibody MA1-80055. Detection of HSC70 served as loading control. Jurkat cells were used as positive control for Tn antigen expression. c Expression of Tn antigen and aberrant O-glycans in COSMC knockdown cells. Western blot analysis showed a strong expression of aberrant O-glycans as well as Tn antigen in Panc-1 COSMC knockdown cells compared to control cells. Sialyl-Tn and Tn antibodies were used as well as lectins such as VVL (Vicia villosa lectin) and WFL (Wisteria floribunda lectin)

Mentions: The Tn antigen (GalNAcα1-O-Ser/Thr) is a frequently occurring aberrant O-glycan expressed at high levels in many cancers [5] including pancreatic ductal adenocarcinoma (PDAC) [6–8], PDAC precursor lesions [7] and is detectable in PDAC sera [9, 10]. Expression of Tn antigen and its sialylated form sialyl-Tn (STn) antigen is associated with poor survival [11, 12] and promotes oncogenic features [4]. Tn antigen expression is initiated by polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), which connect GalNAc residues with the target protein as a posttranslational modification. In humans, 20 different GalNAc-Ts are identified so far, leading to a complex interplay of various enzymes reflected in the cellular O-glycobiome. Tn antigen is further processed by core 1 synthase (C1GALT1 or T-synthase), which transfers Galactose (Gal) to GalNAc-Ser/Thr to form the T antigen, also referred as core 1 structure. COSMC (C1GALT1C1) is the unique chaperone of T-synthase and is essential for its functional formation in order to elongate glycans beyond the initial Tn structure (Fig. 1a) [13]. Dysfunctional COSMC is also able to convert a wild type protein into a tumor-specific antigen [14] affecting tumor cell biology.Fig. 1


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)

Expression of aberrant O-glycans in pancreatic cancer. a Biosynthesis of Tn antigen, sTn antigen and Core1 and 3 structures. Tn antigen is composed of an O-glycosidic linked N-acetylgalactosamine (GalNAc) to the –OH group of serine/threonine (S/T). Tn antigen is either processed by core 1 T-synthase (C1GalT1) and its chaperone (COSMC), which transfers a galactose (Gal) to GalNAc-serine/threonine to form the T antigen also referred as core 1 structure or processed by transfer of a N-acetylglucosamine (GlcNAc) to form the core 3 structure. Tn antigen can also be modified by addition of a sialic acid (NeuAc). b Differential expression of Tn antigen in pancreatic carcinoma cell lines. Eight different PDAC cell lines were available for analysis. Western and Far-Western blot analysis of total cell lysates was performed using the Tn antigen specific antibody MA1-80055. Detection of HSC70 served as loading control. Jurkat cells were used as positive control for Tn antigen expression. c Expression of Tn antigen and aberrant O-glycans in COSMC knockdown cells. Western blot analysis showed a strong expression of aberrant O-glycans as well as Tn antigen in Panc-1 COSMC knockdown cells compared to control cells. Sialyl-Tn and Tn antibodies were used as well as lectins such as VVL (Vicia villosa lectin) and WFL (Wisteria floribunda lectin)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4447007&req=5

Fig1: Expression of aberrant O-glycans in pancreatic cancer. a Biosynthesis of Tn antigen, sTn antigen and Core1 and 3 structures. Tn antigen is composed of an O-glycosidic linked N-acetylgalactosamine (GalNAc) to the –OH group of serine/threonine (S/T). Tn antigen is either processed by core 1 T-synthase (C1GalT1) and its chaperone (COSMC), which transfers a galactose (Gal) to GalNAc-serine/threonine to form the T antigen also referred as core 1 structure or processed by transfer of a N-acetylglucosamine (GlcNAc) to form the core 3 structure. Tn antigen can also be modified by addition of a sialic acid (NeuAc). b Differential expression of Tn antigen in pancreatic carcinoma cell lines. Eight different PDAC cell lines were available for analysis. Western and Far-Western blot analysis of total cell lysates was performed using the Tn antigen specific antibody MA1-80055. Detection of HSC70 served as loading control. Jurkat cells were used as positive control for Tn antigen expression. c Expression of Tn antigen and aberrant O-glycans in COSMC knockdown cells. Western blot analysis showed a strong expression of aberrant O-glycans as well as Tn antigen in Panc-1 COSMC knockdown cells compared to control cells. Sialyl-Tn and Tn antibodies were used as well as lectins such as VVL (Vicia villosa lectin) and WFL (Wisteria floribunda lectin)
Mentions: The Tn antigen (GalNAcα1-O-Ser/Thr) is a frequently occurring aberrant O-glycan expressed at high levels in many cancers [5] including pancreatic ductal adenocarcinoma (PDAC) [6–8], PDAC precursor lesions [7] and is detectable in PDAC sera [9, 10]. Expression of Tn antigen and its sialylated form sialyl-Tn (STn) antigen is associated with poor survival [11, 12] and promotes oncogenic features [4]. Tn antigen expression is initiated by polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), which connect GalNAc residues with the target protein as a posttranslational modification. In humans, 20 different GalNAc-Ts are identified so far, leading to a complex interplay of various enzymes reflected in the cellular O-glycobiome. Tn antigen is further processed by core 1 synthase (C1GALT1 or T-synthase), which transfers Galactose (Gal) to GalNAc-Ser/Thr to form the T antigen, also referred as core 1 structure. COSMC (C1GALT1C1) is the unique chaperone of T-synthase and is essential for its functional formation in order to elongate glycans beyond the initial Tn structure (Fig. 1a) [13]. Dysfunctional COSMC is also able to convert a wild type protein into a tumor-specific antigen [14] affecting tumor cell biology.Fig. 1

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