Abstract 5801: Loss of core 1 β1,3-galactosyltransferase (C1GALT1) in pancreatic cancer leads to altered mucin glycosylation and increased tumor aggressiveness

Abstract

Abstract Background: Aberrant expression of mucins underlie pancreatic cancer (PC) progression and metastasis. Mucin-type O-glycosylation is the principal post-translational modification on mucins and is regulated by myriad of glycosyltransferases. Not much has been investigated about the specific glycosyltransferases involved in increased PC aggressiveness. We found that core 1 β1,3-galactosyltransferase (C1GALT1), which is essential for O-glycosylation, is not expressed in some of the PC patients. C1GALT1 adds galactose in O-glycosylation pathway that forms core 1 structure, which is usually elongated to several carbohydrate structures. Loss of C1GALT1 is accompanied by incomplete O-glycosylation that results in increased expression of truncated carbohydrate antigens (Tn and sTn). Increased expression of these truncated carbohydrate antigens on mucins has been associated with augmented aggressiveness in several malignancies. Based on these results, we hypothesized that loss of C1GALT1 in PC patients is associated with altered mucin O-glycosylation and increased aggressiveness. Methods: Expression of C1GALT1 was examined in HPNE (human pancreatic nestin expressing cells) and oncogenic transformed HPNE cells. To understand the role of C1GALT1 in PC, CRISPR/Cas9 mediated knockout of CIGALT1 was performed in PC cells. Impact of C1GALT1 knockout on glycosylation profile and mucin glycosylation was evaluated using lectin blotting and lectin-pull down assay. Wound-healing assay was performed to examine the effect of C1GALT1 knockout on migration. To gain mechanistic insight, protein expression of signaling molecules involved in growth and motility was evaluated using western blotting. Orthotopic implantation of C1GALT1 knockout cells was carried in pancreas of nude mice to study its role in tumor growth and metastasis. KPC mouse model (KrasG12D; Trp53R172H/+; Pdx-1- Cre) of PC progression was crossed with C1galt1 floxed mice (resulted in KPCC mice) to study the involvement of C1galt1 in disease progression and metastasis in vivo. Results: Oncogenic transformed HPNE cells showed decreased expression of C1GALT1 as compared to untransformed HPNE cells. Knockout of C1GALT1 in PC cells resulted in increased expression of truncated carbohydrate antigens (Tn and sTn). Further, knockout cells displayed increased cellular protrusions, which was associated with significantly increased migration. Western blotting demonstrated increased expression of proteins involved in growth and motility. Lectin-pull down assay revealed altered MUC16 glycosylation in C1GALT1 knockout cells. In vivo studies using orthotopic implantation and KPCC mouse model demonstrated increased tumor weight and metastasis with knockout of C1GALT1. Conclusion: Overall, our results indicate that loss of C1GALT1 in PC is associated with altered MUC16 glycosylation and increased PC aggressiveness. Citation Format: Seema Chugh, Satyanarayana Rachagani, Xinheng Yu, Sriram Neelamegham, Lijun Xia, Michel M. Ouellette, Moorthy P. Ponnusamy, Surinder K. Batra. Loss of core 1 β1,3-galactosyltransferase (C1GALT1) in pancreatic cancer leads to altered mucin glycosylation and increased tumor aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5801. doi:10.1158/1538-7445.AM2017-5801