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

Abstract

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.

Nucleolin-Sle A Glycoforms as E-Selectin Ligands and Potentially Targetable Biomarkers at the Cell Surface of Gastric Cancer Cells

“…Importantly, NCL-SLeA glycoforms showed significant associations with worst overall survival in GC which were not evident based on NCL evaluation alone. These findings support the importance of targeting specific protein glycoforms in order to improve the cancer-specificity of relevant biomarkers, as previously demonstrated by us for other models [34]. Equally important, NCL-SLeA glycoforms were detected in the primary tumors as well as the corresponding metastases, supporting a possible role in disease dissemination mediated by SLeA-E-selectin interactions [80].…”

“…The bands were also excised from the gels, reduced, alkylated and digested with trypsin and identified by mass spectrometry. Tryptic digestion and nanoLC-nES-MS/MS analysis were carried out according to the conditions previously described [34]. Data was analyzed automatically using the SequestHT search engine with the Percolator algorithm for validation of protein identifications (Proteome Discoverer 1.4, Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Formalin-fixed paraffin embedded (FFPE) GC tissue sections showing distinct clinicopathological features (intestinal and diffuse type GC) and healthy tissues (appendix, skin, lung, pancreas, colon, small intestine, gallbladder, liver, testis, stomach, thyroid and tonsil) were screened by immunohistochemistry for both SLeA and NCL, as previously described by us [34]. Briefly, FFPE tissue sections were deparaffinized with xylene, rehydrated with a graded series of alcohol washes and subjected to heat-induced antigen retrieval using citrate buffer pH = 6.0 (Vector Laboratories, Burlingame, CA, USA) for 15 min in the microwave, after pre-heating of the solution at maximum power rating for 5 min.…”

A Systematic Review on the Implications of O-linked Glycan Branching and Truncating Enzymes on Cancer Progression and Metastasis

“…Moreover, sialyl-Tn expressing cells had pronounced BCG-dependent adhesion, internalization, and apoptosis. To test glycosylation variations in bladder tumors, a novel approach of utilizing cancer cell lines with different genetic backgrounds was tested [90]. Herein, T24, 5367, and HT1376 cells were subjected to hypoxic conditions and they were found to express elevated levels of sialyl-Tn as compared to the normal control.…”

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

“…Many secreted proteins are glycosylated, and secreted glycoproteins in body fluids may serve as excellent noninvasive biomarkers. One of the most important glycoproteomic applications in clinical samples is to discover effective disease biomarkers (Abd Hamid et al, 2008;Kaji et al, 2013;Benicky et al, 2014;Mayampurath et al, 2014;Pompach et al, 2014;Zhang et al, 2014;Song and Mechref, 2015;Cotton et al, 2017;Song et al, 2018). Unlike traditional strategies that screen a single or a small number of potential biomarker(s), monitoring a larger group of candidates using glycoproteomic approaches can often result in higher accuracy and specificity.…”