Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes proteinO-glycosylation extension

Abstract

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.

Targeted O‐glycoproteomics explored increased sialylation and identified MUC16 as a poor prognosis biomarker in advanced‐stage bladder tumours

“…Interestingly, this mimics the sialylation of the Tn antigen, whose biological and clinical significance has been extensively studied by our group. Furthermore, we have again reinforced the association between STn antigen expression and aggressive disease, raising to over 300 the number of evaluated tumour sections of different clinicopathological classifications and aetiologies (Bernardo et al ., ; Cabral et al ., ; Costa et al ., ; Ferreira et al ., ; Lima et al ., ; Peixoto et al ., ; Santos et al ., ). Significant efforts should be put on providing accurate quantification of these antigens using high‐throughput glycomics approaches and on developing highly specific ligands.…”

“…). This list included CD44, a typical bladder cancer stem‐cell associated glycoprotein also associated with drug‐resistant phenotypes and poor prognosis (Kobayashi et al ., ), and several integrins, in accordance with previous observations (Peixoto et al ., ). For validation purposes, we have immunoprecipitated CD44 and ITGB1 in these samples and confirmed the expression of STn by western blot (Fig.…”

“…Possible modulation by secreted galactosidases, sialidases are also a possibility that should be investigated. Noteworthily, we have previously observed that bladder cancer cells exposed to hypoxia, a common microenvironmental feature in advanced tumours, promoted a striking downregulation in C2GnT accompanied by an increase in C1GalT1 (Peixoto et al ., ). It is possible that similar events may account for T antigen accumulation in bladder tumours.…”

“…Reinforcing these notions, we have previously demonstrated that 70% of advanced‐stage bladder tumours express the cancer‐associated carbohydrate antigen sialyl‐Tn (Costa et al ., ; Ferreira et al ., ); conversely, the healthy urothelium and most superficial tumours do not (Ferreira et al ., ). STn expression favours cell invasion, motility (Ferreira et al ., ; Peixoto et al ., ) and immune tolerance (Carrascal et al ., ) and has been associated with poor overall survival (Costa et al ., ). In addition, solid tumours often accumulate the more complex T antigen and its sialylated form ST, whose overexpression has also been associated with poor prognosis (Dow et al ., ; Videira et al ., ).…”

Hypoxia-Induced Degenerative Protein Modifications Associated with Aging and Age-Associated Disorders

“…Zanelli et al [103] tested the impact of hypoxia-induced protein nitration on the distribution of nitrotyrosine-like immunoreactivity in the hippocampus of the guinea pig fetus. Hypoxia-induced modifications were documented and reviewed including Slug SUMOylation [104], acetylation [105], glycosylation [106]. Apart from these protein modifications, this review article focuses on hypoxia-induced non-enzymatic post-translational degenerative protein modifications.…”