Background: Glycosylation is the most common post-translational modification of proteins, and glycosylation changes at cell surfaces are frequently associated with malignant epithelia. These affect many functions including proliferation, motility and invasiveness, increasing the propensity to metastasise. Many head & neck squamous cell carcinomata (HNSCC), especially those originating in the lymphoid mucosa of the oropharynx, are driven by so-called high risk genotypes of Human Papillomavirus (HPV). Five-year survival remains poor, averaging around 50% globally: this is partly related to late diagnosis. Specific protein glycosylation signatures on malignant keratinocytes have promise as diagnostic and prognostic markers and as therapeutic targets. Here we begin creation of a glyco-fingerprint library mapping the glyco-space of HNSCC.

Materials and methods: Six established HNSCC cell lines were used to capture the qualitative and semi-quantitative profile of the N- and O-glycome using the well-established porous graphitized carbon (PGC) glycomics approach. Furthermore, we attempted to evaluate the effect sialylation has on HNSCC cell migration by enzymatically removing sialic acid residues from cell surface glycoconjugates. Therefore, we used the enzyme neuraminidase to remove sialic acids from HNSCC cells and measured cell proliferation rates by using incucyte.

Results: In Human Papillomavirus (HPV) negative HNSCC cell lines, 37-41% of cells carried oligomannoses on their surfaces compared to 45% in HPV+ cell lines. Sialylated N-glycan were the most abundant type of N-glycan, contributing to >40% of the N-glycome in the HPV negative cell lines: but just 32-38% in HPV+ cell lines; di- and tri-sialylated forms were most frequent. The majority of complex type N-glycans (33%) carried core-fucose residues with just minor levels (<3%) of Lewis-type fucosylation identified. We also observed unusual types of oligomannose N-glycans carrying core-fucose residues. Enzymatic sialic acid removal resulted in 40% reduction in cell doubling numbers.

Conclusion: This first systematic mapping reveals diverse glycosylation of head and neck cancer cell lines with high levels of core fucosylation and sialylation. Differences between HPV positive and negative cells may partly explain the comparatively good prognosis of HPV-driven HNC. This mapping forms the basis of histopathological profiling of real tumours which might have prognostic potential and suggest targets for immunotherapy. Removal of sialic acids resulted in decreased cell proliferation in vitro, indicating the fundamental role of protein glycosylation in the pathogenesis of HNC.