Aberrant Glycosylation in Cancer: A Novel Molecular Mechanism Controlling Metastasis

Glycosylation alterations are involved in several steps of human cancer pathogenesis. In this issue of Cancer Cell, Agrawal et al. identified the glycosyltransferase FUT8 as a previously unrecognized mediator of melanoma metastasis, establishing core fucosylation as a potential therapeutic target for prevention and treatment of metastatic tumors.The authors acknowledge funding by FEDER, COMPETE and FCT: POCI-01-0145-FEDER-007274 (UID/BIM/04293/2013), POCI-01-0145-FEDER-016585 (PTDC/BBB-EBI/0567/2014), NORTE 2020 (NORTE-01-0145-FEDER-000029), and EU 7th Framework Programme ITN 316929

Glycoengineered cell models for the characterization of cancer O-glycoproteome: an innovative strategy for biomarker discovery

Glycosylation is one of the most abundant forms of protein posttranslational modification. O-glycosylation is a major type of protein glycosylation, comprising different types and structures expressed in several physiologic and pathologic conditions. The understanding of protein attachment site and glycan structure is of the utmost importance for the clarification of the role glycosylation plays in normal cells and in pathological conditions. Neoplastic transformation frequently shows the expression of immature truncated O-glycans. These aberrantly expressed O-glycans have been shown to induce oncogenic properties and can be detected in premalignant lesions, meaning that they are an important source of biomarkers. This article addresses the recent application of genetically engineered cancer cell models to produce simplified homogenous O-glycans allowing the characterization of cancer cells O-glycoproteomes, using advanced mass spectrometry methods and the identification of potential cancer-specific O-glycosylation sites. This article will also discuss possible applications of these biomarkers in the cancer field.IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. The authors were supported by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT-Foundation for Science and Technology, under the projects: PEst-C/SAU/LA0003/2013 and PTDC/BBB-EBI/0786/2012. D Campos has received a grant from the FCT (grant number: SFRH/BD/73717/2010). D Freitas has received a grant from the FCT (grant number: PD/BI/105913/2014)

Glycosylation of cancer extracellular vesicles: Capture strategies, functional roles and potential clinical applications

Glycans are major constituents of extracellular vesicles (EVs). Alterations in the glycosylation pathway are a common feature of cancer cells, which gives rise to de novo or increased synthesis of particular glycans. Therefore, glycans and glycoproteins have been widely used in the clinic as both stratification and prognosis cancer biomarkers. Interestingly, several of the known tumor-associated glycans have already been identified in cancer EVs, highlighting EV glycosylation as a potential source of circulating cancer biomarkers. These particles are crucial vehicles of cell–cell communication, being able to transfer molecular information and to modulate the recipient cell behavior. The presence of particular glycoconjugates has been described to be important for EV protein sorting, uptake and organ-tropism. Furthermore, specific EV glycans or glycoproteins have been described to be able to distinguish tumor EVs from benign EVs. In this review, the application of EV glycosylation in the development of novel EV detection and capture methodologies is discussed. In addition, we highlight the potential of EV glycosylation in the clinical setting for both cancer biomarker discovery and EV therapeutic delivery strategies.This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors COMPETE 2020 (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274) and national funds through the Foundation for Science and Technology (FCT), under the projects:PTDC/BBB-EBI/0567/2014 to C.A.R and UID/BIM/04293/2013; and the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Complement Decay-Accelerating Factor is a modulator of influenza A virus lung immunopathology

Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that the NA-dependent DAF removal of sialic acids exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads, but rather on the host resilience to infection, and may have direct implications in zoonotic influenza transmissions.This work was funded by Instituto Gulbenkian de Ciência (IGC), Fundac¸ão Calouste Gulbenkian (FCG) and Fundação para a Ciência e a Tecnologia (FCT) (PTDC/IMI-MIC/1142/2012). NBS was funded by Graduate Programme Science for Development (PGCD) and FCG. ZEVS was funded by FCT (SFRH/BD/52179/2013). CG was funded by FCT (POCI-01-0145-FEDER-29780, PTDC/MEDQUI/29780/2017). CAR was funded by FCT (POCI-01-0145-FEDER-007274, UID/BIM/04293). MJA is funded by FCT (2020.02373.CEECIND). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Impact of Truncated O-glycans in Gastric-Cancer-Associated CD44v9 Detection

CD44 variant isoforms are often upregulated in cancer and associated with increased aggressive tumor phenotypes. The CD44v9 is one of the major protein splice variant isoforms expressed in human gastrointestinal cancer cells. Immunodetection of CD44 isoforms like CD44v9 in tumor tissue is almost exclusively performed by using specific monoclonal antibodies. However, the structural variability conferred by both the alternative splicing and CD44 protein glycosylation is disregarded. In the present work, we have evaluated the role of O-glycosylation using glycoengineered gastric cancer models in the detection of CD44v9 by monoclonal antibodies. We demonstrated, using different technical approaches, that the presence of immature O-glycan structures, such as Tn and STn, enhance CD44v9 protein detection. These findings can have significant implications in clinical applications mainly at the detection and targeting of this cancer-related CD44v9 isoform and highlight the utmost importance of considering glycan structures in cancer biomarker detection and in therapy targeting.This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE, grant numbers POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; OCI-01-0145-FEDER-031028; and national funds through the Foundation for Science and Technology (FCT), grant numbers PTDC/BBB-EBI/0567/2014 (to CAR), UID/BIM/04293/2013, and PTDC/MED-QUI/29780/2017; and the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). F. Pinto received a fellowship from FCT (SFRH/BPD/115730/2016)

Glycomic Approaches for the Discovery of Targets in Gastrointestinal Cancer

Gastrointestinal (GI) cancer is the most common group of malignancies and many of its types are among the most deadly. Various glycoconjugates have been used in clinical practice as serum biomarker for several GI tumors, however, with limited diagnose application. Despite the good accessibility by endoscopy of many GI organs, the lack of reliable serum biomarkers often leads to late diagnosis of malignancy and consequently low 5-year survival rates. Recent advances in analytical techniques have provided novel glycoproteomic and glycomic data and generated functional information and putative biomarker targets in oncology. Glycosylation alterations have been demonstrated in a series of glycoconjugates (glycoproteins, proteoglycans, and glycosphingolipids) that are involved in cancer cell adhesion, signaling, invasion, and metastasis formation. In this review, we present an overview on the major glycosylation alterations in GI cancer and the current serological biomarkers used in the clinical oncology setting. We further describe recent glycomic studies in GI cancer, namely gastric, colorectal, and pancreatic cancer. Moreover, we discuss the role of glycosylation as a modulator of the function of several key players in cancer cell biology. Finally, we address several state-of-the-art techniques currently applied in this field, such as glycomic and glycoproteomic analyses, the application of glycoengineered cell line models, microarray and proximity ligation assay, and imaging mass spectrometry, and provide an outlook to future perspectives and clinical applications.We acknowledge the support from the European Union, Seventh Framework Programme, Gastric Glyco Explorer initial training network: grant number 316929. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (FCOMP-01-0124- FEDER028188) and National Funds through the FCT-Foundation for Science and Technology, under the projects: PEst-C/SAU/ LA0003/2013, PTDC/BBB-EBI/0786/2012, and PTDC/BBBEBI/0567/2014. AM acknowledges the grant received from FCT, POPH (Programa Operacional Potencial Humano), and FSE (Fundo Social Europeu) (SFRH/BPD/75871/2011). MB acknowledges the University of Girona for pre-doctoral fellowship

Glycosylation in cancer: Mechanisms and clinical implications

Despite recent progress in understanding the cancer genome, there is still a relative delay in understanding the full aspects of the glycome and glycoproteome of cancer. Glycobiology has been instrumental in relevant discoveries in various biological and medical fields, and has contributed to the deciphering of several human diseases. Glycans are involved in fundamental molecular and cell biology processes occurring in cancer, such as cell signalling and communication, tumour cell dissociation and invasion, cell-matrix interactions, tumour angiogenesis, immune modulation and metastasis formation. The roles of glycans in cancer have been highlighted by the fact that alterations in glycosylation regulate the development and progression of cancer, serving as important biomarkers and providing a set of specific targets for therapeutic intervention. This Review discusses the role of glycans in fundamental mechanisms controlling cancer development and progression, and their applications in oncology.The Institute of Molecular Pathology and Immunology of the University of Porto integrates the Institute for Research and Innovation in Health, which is partially supported by the Portuguese Foundation for Science and Technology (FCT). This work is funded by the European Regional Development Fund (FEDER) through the Operational Programme for Competitiveness Factors (COMPETE) and by national funds through the FCT, under the projects PEst‑C/SAU/ LA0003/2013, PTDC/BBB-EBI/0786/2012 and EXPL/BIM-MEC/0149/2012. S.S.P. acknowledges a grant from the FCT (number SFRH/BPD/63094/2009). C.A.R. acknowledges sup­port from the European Union Seventh Framework Programme GastricGlycoExplorer (grant number 316929). The authors apologize that they cannot include all the relevant studies on glycosylation in cancer in this article owing to limitation of space. The authors thank Tiago Fontes- Oliveira for support in figures preparations

The extracellular small leucine-rich proteoglycan biglycan is a key player in gastric cancer aggressiveness

Biglycan (BGN gene), an extracellular proteoglycan, has been described to be associated with cancer aggressiveness. The purpose of this study was to clarify the clinical value of biglycan as a biomarker in multiple independent GC cohorts and determine the in vitro and in vivo role of biglycan in GC malignant features. We found that BGN is commonly over-expressed in all analyzed cohorts, being associated with disease relapse and poor prognosis in patients with advanced stages of disease. In vitro and in vivo experiments demonstrated that biglycan knock-out GC cells display major phenotypic changes with a lower cell survival, migration, and angiogenic potential when compared with biglycan expressing cells. Biglycan KO GC cells present increased levels of PARP1 and caspase-3 cleavage and a decreased expression of mesenchymal markers. Importantly, biglycan deficient GC cells that were supplemented with exogenous biglycan were able to restore biological features, such as survival, clonogenic and migratory capacities. Our in vitro and in vivo findings were validated in human GC samples, where BGN expression was associated with several oncogenic gene signatures that were associated with apoptosis, cell migration, invasion, and angiogenesis. This study provided new insights on biglycan role in GC that should be taken in consideration as a key cellular regulator with major impact in tumor progression and patients’ clinical outcome.This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-029780; POCI-01-0145-FEDER-007274; POCI-01-0145-FEDER-029780) and National Funds through the Foundation for Science and Technology (FCT), under the projects: PTDC/BBB-EBI/0567/2014 (to C.A.R.), PTDC/MED-QUI/29780/2017 (to CG), and UID/BIM/04293 supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). F.P. was funded by FCT cofinanced by Fundo Social Europeu-FSE with a grant with reference: SFRH/BPD/115730/2016

Mechanisms of cisplatin resistance and targeting of cancer stem cells: Adding glycosylation to the equation

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.This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT, the Portuguese Foundation for Science and Technology) under the projects with the references FCOMP-01-0124-FEDER 028188 (PTDC/BBB-EBI/0786/2012) and PTDC/BBB-EBI/0567/2014. C.R. acknowledges the support by Gastric Glyco Explorer Initial Training Network (Seventh Framework Programme grant no. 316929). IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, (PEst-C/SAU/LA0003/2013). Grants were received from FCT: SFRH/BPD/111048/2015 to J.A.F and SFRH/BD/111242/2015 to A.P. FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF)