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Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may offer potential to treat tumour hypoxic niches harbouring more malignant cells.

Decades of research have disclosed a plethora of alterations in protein glycosylation that decisively impact in all stages of disease and ultimately contribute to more aggressive cell phenotypes. The biosynthesis of cancer-associated glycans and its reflection in the glycoproteome is driven by microenvironmental cues and these events act synergistically toward disease evolution. Such intricate crosstalk provides the molecular foundations for the activation of relevant oncogenic pathways and leads to functional alterations driving invasion and disease dissemination. However, it also provides an important source of relevant glyco(neo)epitopes holding tremendous potential for clinical intervention. Therefore, we highlight the transversal nature of glycans throughout the currently accepted cancer hallmarks, with emphasis on the crosstalk between glycans and the tumor microenvironment stromal components. Focus is also set on the pressing need to include glycans and glycoconjugates in comprehensive panomics models envisaging molecular-based precision medicine capable of improving patient care. We foresee that this may provide the necessary rationale for more comprehensive studies and molecular-based intervention. [ABSTRACT FROM AUTHOR]

Bladder carcinogenesis and tumour progression is accompanied by profound alterations in protein glycosylation on the cell surface, which may be explored for improving disease management. In a search for prognosis biomarkers and novel therapeutic targets we have screened, using immunohistochemistry, a series of bladder tumours with differing clinicopathology for short-chain O-glycans commonly found in glycoproteins of human solid tumours. These included the Tn and T antigens and their sialylated counterparts sialyl-Tn(STn) and sialyl-T(ST), which are generally associated with poor prognosis. We have also explored the nature of T antigen sialylation, namely the sialyl-3-T(S3T) and sialyl-6-T(S6T) sialoforms, based on combinations of enzymatic treatments. We observed a predominance of sialoglycans over neutral glycoforms (Tn and T antigens) in bladder tumours. In particular, the STn antigen was associated with high-grade disease and muscle invasion, in accordance with our previous observations. The S3T and S6T antigens were detected for the first time in bladder tumours, but not in healthy urothelia, highlighting their cancer-specific nature. These glycans were also overexpressed in advanced lesions, especially in cases showing muscle invasion. Glycoproteomic analyses of advanced bladder tumours based on enzymatic treatments, Vicia villosa lectin-affinity chromatography enrichment and nanoLC-ESI-MS/MS analysis resulted in the identification of several key cancer-associated glycoproteins (MUC16, CD44, integrins) carrying altered glycosylation. Of particular interest were MUC16 STn+-glycoforms, characteristic of ovarian cancers, which were found in a subset of advanced-stage bladder tumours facing the worst prognosis. In summary, significant alterations in the O-glycome and O-glycoproteome of bladder tumours hold promise for the development of novel noninvasive diagnostic tools and targeted therapeutics. Furthermore, abnormal MUC16 glycoforms hold potential as surrogate biomarkers of poor prognosis and unique molecular signatures for designing highly specific targeted therapeutics. [ABSTRACT FROM AUTHOR]

Little is known on the expression of the tumour‐associated carbohydrate antigen sialyl‐Tn (STn), in bladder cancer. We report here that 75% of the high‐grade bladder tumours, presenting elevated proliferation rates and high risk of recurrence/progression expressed STn. However, it was mainly found in non‐proliferative areas of the tumour, namely in cells invading the basal and muscle layers. STn was also found in tumour‐adjacent mucosa, which suggests its dependence on a field effect of the tumour. Furthermore, it was not expressed by the normal urothelium, demonstrating the cancer‐specific nature of this antigen. STn expression correlated with that of sialyltransferase ST6GalNAc.I, its major biosynthetic enzyme. The stable expression of ST6GalNAc.I in the bladder cancer cell line MCR induced STn expression and a concomitant increase of cell motility and invasive capability. Altogether, these results indicate for the first time a link between STn expression and malignancy in bladder cancer. Hence, therapies targeting STn may constitute new treatment approaches for these tumours. Highlights: First comprehensive study on the expression pattern of carbohydrate antigen STn in bladder cancer.STn is expressed by proliferative/invasive tumours with elevated risk of recurrence/progression.STn is expressed in tumour‐adjacent mucosa, underlining a field carcinogenesis effect.Development of a bladder cancer cell model expressing STn, the MCRSTn+.STn enhances motility and invasive capacity, showing its association with malignancy. [ABSTRACT FROM AUTHOR]

Cancer remains a leading cause of death worldwide due to the lack of safer and more effective therapies. Cancer vaccines developed from neoantigens are an emerging strategy to promote protective and therapeutic anti-cancer immune responses. Advances in glycomics and glycoproteomics have unveiled several cancer-specific glycosignatures, holding tremendous potential to foster effective cancer glycovaccines. However, the immunosuppressive nature of tumours poses a major obstacle to vaccine-based immunotherapy. Chemical modification of tumour associated glycans, conjugation with immunogenic carriers and administration in combination with potent immune adjuvants constitute emerging strategies to address this bottleneck. Moreover, novel vaccine vehicles have been optimized to enhance immune responses against otherwise poorly immunogenic cancer epitopes. Nanovehicles have shown increased affinity for antigen presenting cells (APCs) in lymph nodes and tumours, while reducing treatment toxicity. Designs exploiting glycans recognized by APCs have further enhanced the delivery of antigenic payloads, improving glycovaccine's capacity to elicit innate and acquired immune responses. These solutions show potential to reduce tumour burden, while generating immunological memory. Building on this rationale, we provide a comprehensive overview on emerging cancer glycovaccines, emphasizing the potential of nanotechnology in this context. A roadmap towards clinical implementation is also delivered foreseeing advances in glycan-based immunomodulatory cancer medicine. [Display omitted] • Glycans can be explored in anti-cancer vaccines and/or to target APCs. • Glycans conjugation to immunogens is required for effective immune responses. • Glycovaccine co-administration with immune adjuvants is required for anti-cancer efficacy. • Glycoengineering set foregrounds for glycovaccines but clinical translation is still needed. • A roadmap towards glycovaccines' clinical implementation is provided. [ABSTRACT FROM AUTHOR]