Role of O-glycosylation of Calnexin in Cancer Cell Biology

Abstract

Cancers are a leading cause of morbidity and mortality worldwide. A hallmark of cancerous transformation is aberrant O-glycosylation of proteins, specifically increased expression of short and sialylated O-glycans including the Tn antigen (O-GalNAc), yet the mechanisms behind high Tn expression in tumours remain unclear. Previously, a key membrane trafficking event causing high Tn expression was discovered involving the redistribution of O-glycosylation initiation enzymes, polypeptide N-actelygalactosaminyl transferases (GalNAc-Ts), from the Golgi to the endoplasmic reticulum (ER) upon growth factor activation of Golgi-bound Src (Gill et al. 2010). ER-localised GalNAc-Ts have unhindered access to threonine and serine residues, leading to increased modification of ER-trapped and secreted proteins with the Tn antigen. Targeting GalNAc-T2 to the ER was then found to increase MDA-MB-231 breast carcinoma (MDA ER-G2) cell migration and adhesion to the extracellular matrix (ECM) (Gill et al. 2013). Here, expressing ER-localised GalNAc-T2 was also found to increase MDA cell migration and adhesion as well as increasing their capacity to degrade matrix proteins. O-GalNAc mass spectrometry identified the ER-resident protein calnexin as a substrate of ER-localised GalNAc-Ts. Calnexin was confirmed as a Tn-modified protein in MDA ER-G2 cells and also in liver tumour tissue in vivo. Expression of ER-localised GalNAc-T2 in MDA cells resulted in the cell surface expression of calnexin and led to an interaction with a principal cell-ECM receptor, β1 integrin. Depletion of calnexin in MDA ER-G2 cells reduced their adhesive ability and matrix degradation capacity. Finally, expression of a calnexin mutant lacking Tn modification sites was unable to rescue fully the matrix degradation capacity of MDA ER-G2 cells depleted of endogenous calnexin. These findings lead to the conclusion that during tumourigenesis relocation of GalNAc-Ts to the ER results in the modification of calnexin with Tn, causing its translocation to the cell surface to promote cancer cell behaviour, in part through interactions with β1 integrin. Based on previous data, this pathway is likely to be activated in a large proportion of human cancers and is therefore a potential therapeutic target.

Date of Award	1 Aug 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Humphries (Supervisor) & Martin Lowe (Supervisor)