Dissecting the role of glycan-based tumour promotion in breast cancer

Glycans decorate the surface of all living cells and are among the most diverse post-translational modifications. Mediating manifold interactions between cells and their environment, glycans are also heavily altered during tumorigenesis and aberrant protein glycosylation is a hallmark of cancer. The efficacy of current antibody-based reagents for diagnosis and therapy can be enhanced if target glycosylation is considered. However, our capacity to find the right target protein glycoforms is limited, and the molecular details of tumour promotion by glycans and the corresponding glycosyltransferase enzymes are ill-defined. This work will identify key factors of glycosylation-mediated breast cancer progression by profiling the glycosyltransferase isoenzymes GalNAc-T4, T6 and T14. The three enzymes are of significant relevance in several subtypes and stages of breast cancer, but their protein substrates and mechanisms of disease promotion are unclear. We will use a chemical biology strategy termed bump-and-hole engineering to develop gain-of-function reporter systems for all three isoenzymes. The strategy employs click chemistry to identify protein substrates and, by mass spectrometry (MS) glycoproteomics, glycosylation sites on these substrate proteins in an isoenzyme-specific fashion. We will use these reporter systems to profile for the first time the glycosylation sites modified by GalNAc-T4, T6 and T14 in breast cancer cell lines and primary human tissue samples in the presence of all other glycosyltransferases. We will validate our results by methods of biochemistry, cell biology and, by collaboration, in vivo mouse models of disseminated lung metastasis. We will thereby begin to understand the complexity of glycosylation especially in malignant processes. Our results will not only shed light on the details of glycan-based tumour promotion, but also inform future strategies to develop targeted diagnostics and therapeutics.