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.

Transthyretin (TTR) binds Aβ peptide, preventing its deposition and toxicity. TTR is decreased in Alzheimer’s disease (AD) patients. Additionally, AD transgenic mice with only one copy of the TTR gene show increased brain and plasma Aβ levels when compared to AD mice with both copies of the gene, suggesting TTR involvement in brain Aβ efflux and/or peripheral clearance. Here we showed that TTR promotes Aβ internalization and efflux in a human cerebral microvascular endothelial cell line, hCMEC/D3. TTR also stimulated brain-to-blood but not blood-to-brain Aβ permeability in hCMEC/D3, suggesting that TTR interacts directly with Aβ at the blood-brain-barrier. We also observed that TTR crosses the monolayer of cells only in the brain-to-blood direction, as confirmed by in vivo studies, suggesting that TTR can transport Aβ from, but not into the brain. Furthermore, TTR increased Aβ internalization by SAHep cells and by primary hepatocytes from TTR+/+ mice when compared to TTR−/− animals. We propose that TTR-mediated Aβ clearance is through LRP1, as lower receptor expression was found in brains and livers of TTR−/− mice and in cells incubated without TTR. Our results suggest that TTR acts as a carrier of Aβ at the blood-brain-barrier and liver, using LRP1.

Cisplatin-based chemotherapeutic regimens are the most frequently used (neo)adjuvant treatments for the majority of solid tumors. While platinum-based chemotherapeutic regimens have proven effective against highly proliferative malignant tumors, significant relapse and progression rates as well as decreased overall survival are still observed. Currently, it is known that sub-populations of chemoresistant cells share biological properties with cancer stem cells (CSC), which are believed to be responsible for tumor relapse, invasion and ultimately disease dissemination through acquisition of mesenchymal cell traits. In spite of concentrated efforts devoted to decipher the mechanisms underlying CSC chemoresistance and to design targeted therapeutics to these cells, proteomics has failed to unveil molecular signatures capable of distinguishing between malignant and non-malignant stem cells. This has hampered substantial developments in this complex field. Envisaging a novel rationale for an effective therapy, the current review summarizes the main cellular and molecular mechanisms underlying cisplatin resistance and the impact of chemotherapy challenge in CSC selection and clinical outcome. It further emphasizes the growing amount of data supporting a role for protein glycosylation in drug resistance. The dynamic and context-dependent nature of protein glycosylation is also comprehensively discussed, hence highlighting its potentially important role as a biomarker of CSC. As the paradigm of cancer therapeutics shifts towards precision medicine and patient-tailored therapeutics, we bring into focus the need to introduce glycomics and glycoproteomics in holistic pan-omics models, in order to integrate diverse, multimodal and clinically relevant information towards more effective cancer therapeutics.

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.

Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC.

CD44 is a heavily glycosylated membrane receptor playing a key role in cell adhesion, signal transduction and cytoskeleton remodelling. It is also one of the most studied glycoproteins in cancer, frequently explored for stem cell identification, and associated with chemoresistance and metastasis. However, CD44 is a general designation for a large family of splicing variants exhibiting different degrees of glycosylation and, potentially, functionally distinct roles. Moreover, structural diversity associated with ambiguous nomenclature has delayed clinical developments. Herein, we attempt to comprehensively address these aspects and systematize CD44 nomenclature, setting milestones for biomarker discovery. In addition, we support that CD44 may be an important source of cancer neoantigens, most likely resulting from altered splicing and/or glycosylation. The discovery of potentially targetable CD44 (glyco)isoforms will require the combination of glycomics with proteogenomics approaches, exploring customized protein sequence databases generated using genomics and transcriptomics. Nevertheless, the necessary high-throughput analytical and bioinformatics tools are now available to address CD44 role in health and disease.

Background: Gastric cancer (GC) is a major health burden worldwide, with half of patients developing metastases within 5 years after treatment, urging novel biomarkers for diagnosis and efficient therapeutic targeting. Sialyl-Lewis A (SLeA), a terminal glycoepitope of glycoproteins and glycolipids, offers tremendous potential towards this objective. It is rarely expressed in healthy tissues and blood cells, while it is present in highly metastatic cell lines and metastases. SLeA is also involved in E-selectin mediated metastasis, making it an ideal target to control disease dissemination. Methods and Results: To improve cancer specificity, we have explored the SLeA-glycoproteome of six GC cell models, with emphasis on glycoproteins showing affinity for E-selectin. A novel bioinformatics-assisted algorithm identified nucleolin (NCL), a nuclear protein, as a potential targetable biomarker potentially involved in metastasis. Several immunoassays, including Western blot and in situ proximity ligation reinforced the existence of cell surface NCL-SLeA glycoforms in GC. The NCL-SLeA glycophenotype was associated with decreased survival and was not reflected in relevant healthy tissues. Conclusions: NCL-SLeA is a biomarker of poor prognosis in GC holding potential for precise cancer targeting. This is the first report describing SLeA in preferentially nuclear protein, setting a new paradigm for cancer biomarkers discovery and targeted therapies.

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