The role of O-glycosylation in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has a low survival rate and is characterised by a dense stromal reaction, with high levels of extracellular matrix (ECM), that influences disease progression and response to treatment. Aberrant changes in O-glycosylation contribute to PDAC progression and metastasis. Solid tumours express high levels of the Tn antigen, an O-N-acetylgalactosamine (GalNAc) monosaccharide post-translational modification that has been implicated in cancer progression. The redistribution of polypeptide N-acetylgalactosamine transferases (GALNTs) from Golgi-to-ER, in a process called the GALA pathway, is a proposed mechanism driving high Tn expression in cancer. Whether PDAC has active GALA and how this contributes to tumour development is unknown. To analyse GALA in PDAC, histological analysis of pancreatic tissues was performed for Vicia villosa lectin (VVL). Tn expression in tissue was higher in tumour compared to normal tissue. The distribution of Tn throughout the tumour tissue was highly heterogeneous. In addition, Tn levels were high in normal tumour-adjacent tissues and in acinar cells undergoing acinar-to-ductal metaplasia. VVL affinity enrichment and calnexin immunoblotting identified that GALA was active in some PDAC patient-derived xenografts. Tn levels in pancreatic cancer cells (PCCs) were low compared to models of GALA, indicating low activity of GALA in vitro. GALA was modelled in PCCs by expressing ER-localised GALNT1 (ER-G1). Global and cell surface Tn levels were increased with ER-G1 expression and the cells had an increased ability to degrade ECM. GALA O-glycosylation targets were identified by VVL affinity chromatography coupled to LC-MS/MS. One of the top proteins identified was calnexin. Calnexin was confirmed as a Tn-modified protein in GALA PCCs and was found upregulated at the cell surface in GALA cells. Functional assays to inhibit calnexin by siRNA or antibodies resulted in a reduction in ECM degradation. Spheroid growth and invasion assays identified that GALA cells had a higher invasive capacity and increased growth into Matrigel. The use of anti-calnexin antibodies reduced spheroid growth. These findings led to the conclusion that calnexin facilitates ECM degradation in GALA PCCs, which drives spheroid growth and localised invasion. Using an orthotopic implantation model of PDAC, GALA was shown to increase tumour growth and metastasis. Implantation of KPC cells with inhibition of GALA (ER-2Lec) prevented tumour formation, suggesting GALA is required to expand tumour cells in vivo and develop pancreatic tumours. GALA has been implicated in promoting the reduction of disulphide bonds within ECM to drive ECM degradation. GALA PCCs had a higher propensity to reduce disulphide bonds within ECM, resulting in the increased presence of reduced cysteines. The function of calnexin as an oxidoreductase in pancreatic cancer was assessed by antibody blocking and knockout studies. Inhibition of calnexin prevented the reduction of disulphide bonds within ECM. An assay using iodoacetyl tandem mass tags was developed to assess the ECM substrates of GALA cell surface oxidoreductases by LC-MS/MS. This study provides the first evidence for GALA in pancreatic cancer and demonstrates the role of cell surface calnexin in PCC ECM degradation and reduction.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Humphries (Supervisor) & Claus Jorgensen (Supervisor)