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

Cadherins Glycans in Cancer: Sweet Players in a Bitter Process

Cadherins are key components in tissue morphogenesis and architecture, contributing to the establishment of cohesive cell adhesion. Reduced cellular adhesiveness as a result of cadherin dysfunction is a defining feature of cancer. During tumor development and progression, major changes in the glycan repertoire of cancer cells take place, affecting the stability, trafficking, and cell-adhesion properties of cadherins. Importantly, the different glycoforms of cadherins are promising biomarkers, with potential clinical application to improve the management of patients, and constitute targets for the development of new therapies. This review discusses the most recent insights on the impact of glycan structure on the regulation of cadherin function in cancer, and provides a perspective on how cadherin glycans constitute tumor biomarkers and potential therapeutic targets.IPATIMUP integrates the I3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação). This work was financed by FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through the FCT in the framework of the project ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274), PTDC/DTP-PIC/0560/2014, and PTDC/BBB-EBI/0567/2014. S.C. also acknowledges funding from the FCT (SFRH/BD/77386/2011)

High Performance Liquid Chromatography Method for the Determination of Anethole in Rat Plasma

Purpose: To identify and quantify anethole in the essential oil of fruits of Illicium verum Hook (star anise) and in vivo in rat plasma using reverse-phase liquid chromatography.Methods: Anethole was identified in the essential oil of the fruits of Star anise and determined by gas chromatography-tandem mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), ultraviolet visible spectrophotometry (UV-VIS). A simple, sensitive and validated high performance liguid chromatography (HPLC) technique with UV-VIS detection method was developed for the determination of the compound in rat plasma using: methanol-water (85:15, v/v) as mobile phase at a flow rate of 0.2 ml/min Hypersil ODS Thermo (150 mm x 2.1 mm x 3.0 μM) as column with wavelength detection at 259 nm.Results: GC determination showed that anethole in the essential oil of star anise exhibited a retention time of 21.02 min. The validation results for anethole in plasma were satisfactory, with coefficient of determination (R2) of 0.9945 and relative standard deviation of < 3 %. HPLC run time of 4 min with a retention time of 2.73 min was the faster method to determine anethole when compared to a previously reported method which had a run time of 15 min.Conclusion: Anethole in the essential oil of Illicium verum Hook can be identified and determined by GC-MS, NMR and UV-VIS, and a superior HPLC method has been developed for the determination of the compound in rat plasma.Keywords: Anethole, High performance liguid chromatography, Star anise, Essential oil, Rat plasma, Illicium verum Hook

Studying T Cells N-Glycosylation by Imaging Flow Cytometry

Imaging flow cytometry is an emerging imaging technology that combines features of both conventional flow cytometry and fluorescence microscopy allowing quantification of the imaging parameters. The analysis of protein posttranslational modifications by glycosylation using imaging flow cytometry constitutes an important bioimaging tool in the glycobiology field. This technique allows quantification of the glycan fluorescence intensity, co-localization with proteins, and evaluation of the membrane/cytoplasmic expression. In this chapter we provide the guidelines to analyze glycan expression, particularly the ß1,6 GlcNAc branched N-glycans, on the membrane of intestinal T cells from inflammatory bowel disease patients.This work was supported by grants from the Portuguese Foundation for Science and Technology (FCT), project grants (PTDC/DTPPIC/0560/2014; PTDC/BBB-EBI/0786/2012; EXPL/BIMMEC/0149/2012), “financiados no âmbito do Programa Operacional Temático Factores de Competitividade (COMPETE) e comparticipado pelo fundo Comunitário Europeu FEDER,” e do Quadro de Referência Estratégia Nacional QREN. This work was further supported by a Portuguese grant from “Grupo de Estudo da Doença Infl amatória Intestinal” (GEDII). This work had also the fi nantial support of FCT/MEC through National Funds and, when applicable, co-fi nanced by the FEDER via the PT2020 Partnership Agreement under the 4293 Unit I&D. S.S.P. (SFRH/BPD/63094/2009) also acknowledges FCT. A.M.D. PD/BD/105982/2014 also acknowledges FCT and BiotechHealth Doctoral Programme. The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) integrates the Institute for Research and Innovation in Health (I3S), which is partially supported by the Portuguese Foundation for Science and Technology (FCT). Data was acquired at the Bioimaging Center for Biomaterials and Regenerative Therapies (b.IMAGE, INEB, Porto, Portugal)

Dysregulation of T cell receptor N-glycosylation: A molecular mechanism involved in ulcerative colitis

The incidence of inflammatory bowel disease is increasing worldwide and the underlying molecular mechanisms are far from being fully elucidated. Herein, we evaluated the role of N-glycosylation dysregulation in T cells as a key mechanism in the ulcerative colitis (UC) pathogenesis. The evaluation of the branched N-glycosylation levelsandprofile of intestinalTcell receptor (TCR)wereassessedin colonic biopsies fromUCpatientsand healthy controls. Expression alterations of the glycosyltransferase gene MGAT5 were also evaluated. We demonstrated thatUCpatients exhibit a dysregulation ofTCRbranchedN-glycosylationonlamina propriaTlymphocytes. Patients with severe UC showed the most pronounced defect on N-glycan branching in T cells. Moreover, UC patients showed a significant reduction of MGAT5 gene transcription in T lymphocytes. In this study, we disclose for the first time that a deficiency in branched N-glycosylation on TCR due to a reduced MGAT5 gene expression is a new molecular mechanism underlying UC pathogenesis, being a potential novel biomarker with promising clinical and therapeutic applications.This work was supported by grants from the Portuguese Foundation for Science and Technology (FCT), project grants (PTDC/ CVT/111358/2009; PTDC/BBB-EBI/0786/2012; EXPL/ BIM-MEC/0149/2012), ‘financiados no âmbito do Programa Operacional Temático Factores de Competitividade (COMPETE) e comparticipado pelo fundo Comunitário Europeu FEDER’, e do Quadro de Referência Estratégia Nacio-nal QREN. This work was further supported by a portuguese grant from ‘Grupo de Estudo da Doenc¸a Inflamatória Intestinal’ (GEDII). S.S.P. (SFRH/BPD/63094/2009); S.C. (SFRH/BD/ 77386/2011) also acknowledge FCT. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and is partially supported by FCT

SAW: A Method to Identify Splicing Events from RNA-Seq Data Based on Splicing Fingerprints

Splicing event identification is one of the most important issues in the comprehensive analysis of transcription profile. Recent development of next-generation sequencing technology has generated an extensive profile of alternative splicing. However, while many of these splicing events are between exons that are relatively close on genome sequences, reads generated by RNA-Seq are not limited to alternative splicing between close exons but occur in virtually all splicing events. In this work, a novel method, SAW, was proposed for the identification of all splicing events based on short reads from RNA-Seq. It was observed that short reads not in known gene models are actually absent words from known gene sequences. An efficient method to filter and cluster these short reads by fingerprint fragments of splicing events without aligning short reads to genome sequences was developed. Additionally, the possible splicing sites were also determined without alignment against genome sequences. A consensus sequence was then generated for each short read cluster, which was then aligned to the genome sequences. Results demonstrated that this method could identify more than 90% of the known splicing events with a very low false discovery rate, as well as accurately identify, a number of novel splicing events between distant exons

O-mannosylation and N-glycosylation: Two coordinated mechanisms regulating the tumour suppressor functions of E-cadherin in cancer

Dysregulation of tumor suppressor protein E-cadherin is an early molecular event in cancer. O-mannosylation profile of E-cadherin is a newly-described posttranslational modification crucial for its adhesive functions in homeostasis. However, the role of O-mannosyl glycans in E-cadherin-mediated cell adhesion in cancer and their interplay with N-glycans remains largely unknown. We herein demonstrated that human gastric carcinomas exhibiting a non-functional E-cadherin display a reduced expression of O-mannosyl glycans concomitantly with increased modification with branched complex N-glycans. Accordingly, overexpression of MGAT5-mediated branched N-glycans both in gastric cancer cells and transgenic mice models led to a significant decrease of O-mannosyl glycans attached to E-cadherin that was associated with impairment of its tumour suppressive functions. Importantly, overexpression of protein O-mannosyltransferase 2 (POMT2) induced a reduced expression of branched N-glycans which led to a protective effect of E-cadherin biological functions. Overall, our results reveal a newly identified mechanism of (dys)regulation of E-cadherin that occur through the interplay between O-mannosylation and N-glycosylation pathway.IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145- FEDER-007274) and under the projects PTDC/DTP-PIC/0560/2014; PTDC/BBB-EBI/0567/2014]. This work was further supported by SFRH/BD/77386/2011 (SC) and the the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 1036, project 11