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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

COSMC knockdown promotes migration and survival and inhibits proliferation in PDAC cells in vitro a Proliferation of Panc-1 COSMC knockdown cells compared to control. Proliferation was measured in MTT assay over 96 h using Panc-1 COSMC knockdown cells (black) and control cells (grey) (p < 0.001). L3.6pl COSMC knockdown and control cells are indicated as dotted line. b above: Scratch assay are displayed for Panc-1 COSMC knockdown cells compared to control at different time points. b below: HE stained transwell membranes after 24 h of Panc-1 COSMC knockdown cells (left) and control cells (right) c: Relative migration of Panc-1 and L3.6pl COSMC knockdown cells was compared to control cells using transwell assay. Migrated cells were determined after 24 h (p ≤ 0.001). d: Apoptosis rate of Panc-1 and L3.6pl COSMC knockdown cells was compared to control cells using ELISA based cleaved Caspase-3 assay. Cleaved Caspase-3 expression was determined after 24 h (p = 0.03 and 0 = 0.017)
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Fig4: COSMC knockdown promotes migration and survival and inhibits proliferation in PDAC cells in vitro a Proliferation of Panc-1 COSMC knockdown cells compared to control. Proliferation was measured in MTT assay over 96 h using Panc-1 COSMC knockdown cells (black) and control cells (grey) (p < 0.001). L3.6pl COSMC knockdown and control cells are indicated as dotted line. b above: Scratch assay are displayed for Panc-1 COSMC knockdown cells compared to control at different time points. b below: HE stained transwell membranes after 24 h of Panc-1 COSMC knockdown cells (left) and control cells (right) c: Relative migration of Panc-1 and L3.6pl COSMC knockdown cells was compared to control cells using transwell assay. Migrated cells were determined after 24 h (p ≤ 0.001). d: Apoptosis rate of Panc-1 and L3.6pl COSMC knockdown cells was compared to control cells using ELISA based cleaved Caspase-3 assay. Cleaved Caspase-3 expression was determined after 24 h (p = 0.03 and 0 = 0.017)

Mentions: To gain relevant insights into COSMC function in pancreatic cancer cells, we first analyzed whether COSMC might influence proliferation of Panc-1 cells. Interestingly, analysis of Panc-1 COSMC knockdown cells by MTT assay revealed a strong reduction in proliferation, compared to control cells (p <0.001), whereas COSMC knockdown had no effect on proliferation in L3.6pl cells (Fig. 4a). Next, we analyzed the influence of COSMC expression on cancer cell migration in vitro. Therefore, we performed transwell assays and quantified the migratory potential of COSMC knockdown as well as corresponding control cells after 24 h. Migration of Panc-1 COSMC knockdown cells was substantially higher compared to control cells (Fig. 4b-c) (p < 0.001). Additionally, migrating cells were recorded in a wound healing assay and the cell displacement rate during migration was quantified. The median displacement rate of Panc-1 COSMC knockdown cells was 154.4 μm/s whereas control cells showed a median displacement rate of 108.1 μm/s (p <0.05). The displacement rate of Panc-1 COSMC knockdown cells was 0.00153 μm/s and that of Panc-1 control cells was 0.00095 μm/s (p <0.05) (representative images are shown in Fig. 4b). The meandering index of Panc-1 COSMC knockdown cells was 0.261 and 0.196 in control cells (p <0.001). To analyze whether COSMC knockdown influences apoptosis in pancreatic cancer, a cleaved Caspase-3 ELISA assay was performed. Interestingly, COSMC depleted Panc-1 and L3.6pl cells showed substantially decreased apoptosis compared to corresponding control cells. Cleaved Caspase-3 expression was determined after 24 h and showed a 53 % reduced apoptosis rate in Panc-1 COSMC knockdown cells (p =0.03). L3.6pl COSMC knockdown cells showed a 44 % reduction of apoptosis rate (p =0.017) compared to control (Fig. 4d).Fig. 4


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)

COSMC knockdown promotes migration and survival and inhibits proliferation in PDAC cells in vitro a Proliferation of Panc-1 COSMC knockdown cells compared to control. Proliferation was measured in MTT assay over 96 h using Panc-1 COSMC knockdown cells (black) and control cells (grey) (p < 0.001). L3.6pl COSMC knockdown and control cells are indicated as dotted line. b above: Scratch assay are displayed for Panc-1 COSMC knockdown cells compared to control at different time points. b below: HE stained transwell membranes after 24 h of Panc-1 COSMC knockdown cells (left) and control cells (right) c: Relative migration of Panc-1 and L3.6pl COSMC knockdown cells was compared to control cells using transwell assay. Migrated cells were determined after 24 h (p ≤ 0.001). d: Apoptosis rate of Panc-1 and L3.6pl COSMC knockdown cells was compared to control cells using ELISA based cleaved Caspase-3 assay. Cleaved Caspase-3 expression was determined after 24 h (p = 0.03 and 0 = 0.017)
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Related In: Results  -  Collection

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Fig4: COSMC knockdown promotes migration and survival and inhibits proliferation in PDAC cells in vitro a Proliferation of Panc-1 COSMC knockdown cells compared to control. Proliferation was measured in MTT assay over 96 h using Panc-1 COSMC knockdown cells (black) and control cells (grey) (p < 0.001). L3.6pl COSMC knockdown and control cells are indicated as dotted line. b above: Scratch assay are displayed for Panc-1 COSMC knockdown cells compared to control at different time points. b below: HE stained transwell membranes after 24 h of Panc-1 COSMC knockdown cells (left) and control cells (right) c: Relative migration of Panc-1 and L3.6pl COSMC knockdown cells was compared to control cells using transwell assay. Migrated cells were determined after 24 h (p ≤ 0.001). d: Apoptosis rate of Panc-1 and L3.6pl COSMC knockdown cells was compared to control cells using ELISA based cleaved Caspase-3 assay. Cleaved Caspase-3 expression was determined after 24 h (p = 0.03 and 0 = 0.017)
Mentions: To gain relevant insights into COSMC function in pancreatic cancer cells, we first analyzed whether COSMC might influence proliferation of Panc-1 cells. Interestingly, analysis of Panc-1 COSMC knockdown cells by MTT assay revealed a strong reduction in proliferation, compared to control cells (p <0.001), whereas COSMC knockdown had no effect on proliferation in L3.6pl cells (Fig. 4a). Next, we analyzed the influence of COSMC expression on cancer cell migration in vitro. Therefore, we performed transwell assays and quantified the migratory potential of COSMC knockdown as well as corresponding control cells after 24 h. Migration of Panc-1 COSMC knockdown cells was substantially higher compared to control cells (Fig. 4b-c) (p < 0.001). Additionally, migrating cells were recorded in a wound healing assay and the cell displacement rate during migration was quantified. The median displacement rate of Panc-1 COSMC knockdown cells was 154.4 μm/s whereas control cells showed a median displacement rate of 108.1 μm/s (p <0.05). The displacement rate of Panc-1 COSMC knockdown cells was 0.00153 μm/s and that of Panc-1 control cells was 0.00095 μm/s (p <0.05) (representative images are shown in Fig. 4b). The meandering index of Panc-1 COSMC knockdown cells was 0.261 and 0.196 in control cells (p <0.001). To analyze whether COSMC knockdown influences apoptosis in pancreatic cancer, a cleaved Caspase-3 ELISA assay was performed. Interestingly, COSMC depleted Panc-1 and L3.6pl cells showed substantially decreased apoptosis compared to corresponding control cells. Cleaved Caspase-3 expression was determined after 24 h and showed a 53 % reduced apoptosis rate in Panc-1 COSMC knockdown cells (p =0.03). L3.6pl COSMC knockdown cells showed a 44 % reduction of apoptosis rate (p =0.017) compared to control (Fig. 4d).Fig. 4

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