Aberrant O-GaINAc glycosylation to enhance AKT/mTOR signaling in pancreatic cancer.

Abstract

Background: Immature truncated O-GalNAc glycosylation is an important feature of pancreatic ductal adenocarcinomas and is detected with high frequency in premalignant lesions. Differential expression of O-GalNAc glycans Tn antigen and sialyl-Tn antigen is strongly associated with decreased survival and poor prognosis and frequently occurs in pancreatic cancer due to hypermethylation of the COSMC (C1GALT1C1)gene. COSMC knockdown experiments displayed reduced rate of apoptosis and enhanced migratory behavior, thereby promoting oncogenic properties in pancreatic cancer cells. Since the underlying biological processes are not well understood, we investigated the impact of aberrant O-GalNAc glycosylation on AKT/mTOR signaling in Panc-1 and L3.6pl COSMC knockdown cells. Methods: Lentiviral mediated COSMC knockdown cell lines were subjected to stable isotope labeling by amino acids (SILAC) and subsequently analyzed by mass spectromic quantitative proteomics. Immunoprecipitation, Western blot analysis and real time PCR were used to assess glycotype dependent signaling molecule expression and phosphorylation status. Results: In COSMC knockdown cells, quantitative proteomics identified proteins associated to IGF, EGF and PI3K pathways. We could further identify AKT as novel O-GalNAc-modified protein in Panc-1 pancreatic cancer cell line. AKT downstream substrates S6 ribosomal protein and GSK-3b displayed an enhanced phosphorylation and AKT upstream effectors, such as IGF-I receptor and mTORC2 complex showed an enhanced activation in COSMC knockdown cells. Interestingly, aberrant O-glycosylation was able to modify mTOR S2448 phosphorylation in L3.6pl COSMC knockdown cells treated with mTORC inhibitor AZD8055. Conclusions: Our study revealed an enhanced AKT/mTOR signaling in pancreatic cancer COSMC knockdown cells, which is driven by aberrant O-GalNAc glycosylation and substantiates the previously observed enhancement of oncogenic properties