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

// Andreia Peixoto 1, 2, 3, 4, * , Elisabete Fernandes 1, 3, 4, 5, * , Cristiana Gaiteiro 1, * , Luis Lima 1, 3, 6 , Rita Azevedo 1, 4 , Janine Soares 1 , Sofia Cotton 1 , Beatriz Parreira 1 , Manuel Neves 1, 4 , Teresina Amaro 7 , Ana Tavares 1, 7 , Filipe Teixeira 8 , Carlos Palmeira 1, 9 , Maria Rangel 10 , Andre M.N. Silva 11 , Celso A. Reis 3, 4, 6, 12 , Lucio Lara Santos 1, 9, 13 , Maria Jose Oliveira 2, 3 , Jose Alexandre Ferreira 1, 3, 4, 6, 14 1 Experimental Pathology and Therapeutics Group, IPO Porto Research Center (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal 2 New Therapies Group, INEB-Institute for Biomedical Engineering, Porto, Portugal 3 Instituto de Investigacao e Inovacao em Saude, Universidade do Porto, Portugal 4 Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal 5 Biomaterials for Multistage Drug and Cell Delivery, INEB-Institute for Biomedical Engineering, Porto, Portugal 6 Glycobiology in Cancer, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal 7 Department of Pathology, Hospital Pedro Hispano, Matosinhos, Portugal 8 LAQV-REQUIMTE, Faculty of Sciences of the University of Porto, Porto, Portugal 9 Health School of University Fernando Pessoa, Porto, Portugal 10 UCIBIO-REQUIMTE, Instituto de Ciencias Biomedicas Abel Salazar, University of Porto, Porto, Portugal 11 UCIBIO-REQUIMTE/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto, Portugal 12 Department of Pathology and Oncology, Faculty of Medicine, Porto University, Porto, Portugal 13 Department of Surgical Oncology, Portuguese Institute of Oncology, Porto, Portugal 14 Porto Comprehensive Cancer Center (P.ccc), Porto, Portugal * These authors contributed equally to this work Correspondence to: Jose Alexandre Ferreira, email: josef@ipatimup.pt Keywords: glycosylation, bladder cancer, hypoxia, invasion, sialyl-Tn Received: September 24, 2015      Accepted: July 26, 2016      Published: August 12, 2016 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.