RESUMO
Truncated O-GalNAc glycosylation is an important feature of pancreatic ductal adenocarcinomas (PDAC) and expression of truncated O-GalNAc glycans is strongly associated with decreased survival and poor prognosis. It has been proven, that aberrant O-GalNAc glycosylation influence PDAC signaling to promote oncogenic properties, but elucidation of the influence of truncated O-GalNAc glycosylation on different signaling molecules has just been started. We herein elucidated the impact of aberrant O-GalNAc glycosylation on two important PDAC signaling pathways, namely AKT/mTOR and RAS/MAPK. In PDAC cells expressing truncated O-GalNAc glycans, we identified differentially expressed proteins associated with AKT/mTOR and RAS/MAPK pathways using quantitative proteomics. Since AKT, a key-signaling molecule in PDAC, was among the identified proteins, we analyzed AKT and found a strikingly enhanced S473 phosphorylation and identified a previously unknown O-GalNAc-modification. Consecutive analysis of COSMC knockdowns in PDAC revealed strong effects on AKT upstream and downstream effector molecules. Interestingly, truncated O-GalNAc glycans could facilitate an mTORC1 inhibitor resistance using AZD8055. In addition, as AKT/mTOR pathway has extensive cross talks with RAS/MAPK pathway we analyzed the pathways and found it negatively regulated. Finally, we found that the expression of epithelial-mesenchymal-transition markers, key features of aggressive PDACs cells, are enhanced and truncated O-GalNAc glycans enhance pancreatic cancer cell growth in a xenograft mouse model. Our study demonstrates that truncated O-GalNAc glycans have a strong impact on AKT/mTOR and RAS/MAPK signaling pathways, are modulated by EGF or IGF-1 signaling and should be considered for targeted therapy of these pathways in PDAC.
RESUMO
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.