Exostosin‐like 2 Abrogation Promotes Heparan Sulfate Biosynthesis and Switches Cancer Cell Signalling Towards an Invasive Phenotype

Abstract

Heparan Sulfate ProteoGlycans (HSPGs) are essential components of cells’ glycocalyx and Extracellular Matrix (ECM), which play key roles in cell physiology and in pathological scenarios. By binding to multiple biological ligands, via HS glycosaminoglycan (GAG) chains, HSPGs modulate cancer cells’ interaction with ECM and signalling networks, ultimately controlling tumour microenvironment and disease development. The aim of this work was to disclose the regulatory mechanisms underlying HSPGs biosynthetic pathways in gastric cancer context. Furthermore, we evaluated the impact of cellular GAGs content in cancer cell signalling and motility features. We developed glycoengineered gastric cancer cell models lacking specific glycosyltransferases involved in HS biosynthesis, Exostosin Like Glycosyltransferase 2 (EXTL2) and EXTL3. Biochemical characterization revealed that abrogation of EXTL2 or EXTL3 impacts both HS and Chondroitin Sulfate (CS) cell levels, as well as GAGs structural conformations, supporting that both enzymes exert broad regulatory roles in GAGs biosynthesis pathways. We observed that while EXTL3 is key for initiating the synthesis of HS chains, in detriment of CS biosynthesis, EXTL2 functions as a negative regulator of HS biosynthesis. Gene expression and protein levels of two major HS carriers were also determined and revealed that EXTL2 KO promoted significant alterations in HSPG core protein levels. Furthermore, we addressed the effects of aberrant GAGosylation over tumour cells motility and signalling events. Cellular functional analysis demonstrated that EXTL2 KO and concomitant HS increase promoted motile and invasive phenotypes, as well as the activation of key cell surface receptor tyrosine kinase. Overall, our results reveal the crucial roles of EXTL2 and EXTL3 in the modulation of proteoglycans expression and aberrant glycosylation profiles displayed by cancer cells, and the functional impact of these alterations on cells malignant behaviour, further supporting the clinical potential of HS biosynthetic machinery in cancer therapy.