Salomé S. Pinho

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.

Epithelial E- and P-cadherins: Role and clinical significance in cancer

E-cadherin and P-cadherin are major contributors to cell-cell adhesion in epithelial tissues, playing pivotal roles in important morphogenetic and differentiation processes during development, and in maintaining integrity and homeostasis in adult tissues. It is now generally accepted that alterations in these two molecules are observed during tumour progression of most carcinomas. Genetic or epigenetic alterations in E- and P-cadherin-encoding genes (CDH1 and CDH3, respectively), or alterations in their proteins expression, often result in tissue disorder, cellular de-differentiation, increased invasiveness of tumour cells and ultimately in metastasis. In this review, we will discuss the major properties of E- and P-cadherin molecules, its regulation in normal tissue, and their alterations and role in cancer, with a specific focus on gastric and breast cancer models.

Modulation of E-cadherin function and dysfunction by N -glycosylation

Several mechanisms have been proposed to explain the E-cadherin dysfunction in cancer, including genetic and epigenetic alterations. Nevertheless, a significant number of human carcinomas have been seen that show E-cadherin dysfunction that cannot be explained at the genetic/epigenetic level. A substantial body of evidence has appeared recently that supports the view that other mechanisms operating at the post-translational level may also affect E-cadherin function. The present review addresses molecular aspects related to E-cadherin N-glycosylation and evidence is presented showing that the modification of N-linked glycans on E-cadherin can affect the adhesive function of this adhesion molecule. The role of glycosyltransferases involved in the remodeling of N-glycans on E-cadherin, including N-acetylglucosaminyltransferase III (GnT-III), N-acetylglucosaminyltransferase V (GnT-V), and the α1,6 fucosyltransferase (FUT8) enzyme, is also discussed. Finally, this review discusses an alternative functional regulatory mechanism for E-cadherin operating at the post-translational level, N-glycosylation, that may underlie the E-cadherin dysfunction in some carcinomas.

The role of N-acetylglucosaminyltransferase III and V in the post-transcriptional modifications of E-cadherin

It has long been recognized that E-cadherin dysfunction is a major cause of epithelial cell invasion. However, very little is known about the post-transcriptional modifications of E-cadherin and its role in E-cadherin mediated tumor progression. N-acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, and has been pointed as a metastasis suppressor. N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6 GlcNAc branching of N-glycans, and has been associated to increase metastasis. The regulatory mechanism between E-cadherin expression and the remodeling of its oligosaccharides structures by GnT-III and GnT-V were explored in this study. We have demonstrated that wild-type E-cadherin regulates MGAT3 gene transcription resulting in increased GnT-III expression. We also showed that GnT-III and GnT-V competitively modified E-cadherin N-glycans. The GnT-III knockdown cells revealed a membrane de-localization of E-cadherin leading to its cytoplasmic accumulation. Further, the GnT-III knockdown cells also caused modifications of E-cadherin N-glycans catalyzed by GnT-III and GnT-V. Altogether our results have clarified the existence of a bidirectional crosstalk between E-cadherin and GnT-III/GnT-V that was, for the first time, reproduced in an in vivo model. This study opens new insights into the post-transcriptional modifications of E-cadherin in its biological function, in a tumor context.

Loss and Recovery of Mgat3 and GnT-III Mediated E-cadherin N-glycosylation Is a Mechanism Involved in Epithelial-Mesenchymal-Epithelial Transitions

Background N-acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase encoded by Mgat3 that catalyzes the addition of β1,4-bisecting-N-acetylglucosamine on N-glycans. GnT-III has been pointed as a metastases suppressor having varying effects on cell adhesion and migration. We have previously described the existence of a functional feedback loop between E-cadherin expression and GnT-III-mediated glycosylation. The effects of GnT-III-mediated glycosylation on E-cadherin expression and cellular phenotype lead us to evaluate Mgat3 and GnT-III-glycosylation role during Epithelial-Mesenchymal-Transition (EMT) and the reverted process, Mesenchymal-Epithelial-Transition (MET). Methodology/Principal Findings We analyzed the expression profile and genetic mechanism controlling Mgat3 expression as well as GnT-III-mediated glycosylation, in general and specifically on E-cadherin, during EMT/MET. We found that during EMT, Mgat3 expression was dramatically decreased and later recovered when cells returned to an epithelial-like phenotype. We further identified that Mgat3 promoter methylation/demethylation is involved in this expression regulation. The impact of Mgat3 expression variation, along EMT/MET, leads to a variation in the expression levels of the enzymatic product of GnT-III (bisecting GlcNAc structures), and more importantly, to the specific modification of E-cadherin glycosylation with bisecting GlcNAc structures. Conclusions/Significance Altogether, this work identifies for the first time Mgat3 glycogene expression and GnT-III-mediated glycosylation, specifically on E-cadherin, as a novel and major component of the EMT/MET mechanism signature, supporting its role during EMT/MET.

Molecular Carcinogenesis of Canine Mammary Tumors News From an Old Disease

Studies focusing on the molecular basis of canine mammary tumors (CMT) have long been hampered by limited numbers of molecular tools specific to the canine species. The lack of molecular information for CMT has impeded the identification of clinically relevant tumor markers beyond histopathology and the introduction of new therapeutic concepts. Additionally, the potential use for the dog as a model for human breast cancer is debatable until questions are answered regarding cellular origin, mechanisms, and cellular pathways. During the past years, increasing numbers of canine molecular tools have been developed on the genomic, RNA, and protein levels, and an increasing number of studies have shed light on specific aspects of canine carcinogenesis, particularly of the mammary gland. This review summarizes current knowledge on the molecular carcinogenesis of CMT, including the role of specific oncogenes, tumor suppressors, regulators of apoptosis and DNA repair, proliferation indices, adhesion molecules, circulating tumor cells, and mediators of angiogenesis in CMT progression and clinical behavior. Whereas the data available are far from complete, knowledge of molecular pathways has a significant potential to complement and refine the current diagnostic and therapeutic approach to this tumor type. Furthermore, current data show that significant similarities and differences exist between canine and human mammary tumors at the molecular level. Clearly, this is only the beginning of an understanding of the molecular mechanisms of CMT and their application in clinical patient management.

Role of E-cadherin N-glycosylation profile in a mammary tumor model.

Modifications in cell surface glycosylation affecting cell adhesion are common characteristics of transformed cells. This study characterizes the N-glycosylation profile of E-cadherin in models of canine mammary gland adenoma and carcinoma evaluating the importance of these glycosylation modifications in the malignant phenotype. Our results show that the pattern of E-cadherin N-glycosylation in mammary carcinoma is characterized by highly branched N-glycans, increase in sialylation and an expression of few high mannose structures. Detailed mass spectrometry analysis demonstrated a new N-glycosylation site containing a potential complex type N-glycan in E-cadherin from a mammary carcinoma cell line. Our study demonstrates the importance of E-cadherin N-glycans in the process of tumor development and in the transformation to the malignant phenotype.

Gastric cancer: adding glycosylation to the equation

Gastric cancer has a high incidence and mortality, so there is a pressing need to understand the underlying molecular mechanisms in order to discover novel biomarkers. Glycosylation alterations are frequent during gastric carcinogenesis and cancer progression. This review describes the role of glycans from the initial steps of the carcinogenesis process, in which Helicobacter pylori adheres to host mucosa glycans and modulates the glycophenotype, as well as how glycans interfere with epithelial cell adhesion by modulating epithelial cadherin functionality in gastric cancer progression. Other mechanisms regulating gastric cancer malignant behavior are discussed, such as increased sialylation interfering with key signaling pathways and integrin glycosylation leading to an invasive phenotype. Applications of these glycosylation alterations in the clinical management of gastric cancer patients are discussed.

Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer

E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell–cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression.

Sialyl Lewis x expression in canine malignant mammary tumours: correlation with clinicopathological features and E-Cadherin expression

BackgroundSialyl Lewis x (sLex) antigen is a carbohydrate antigen that is considered not only a marker for cancer but also implicated functionally in the malignant behaviour of cancer cells. Overexpression of sLex is associated with enhanced progression and metastases of many types of cancer including those of the mammary gland. Canine mammary tumours can invade and give rise to metastases via either lymphatic or blood vessels.E-Cadherin is specifically involved in epithelial cell-to-cell adhesion. In cancer, E-Cadherin underexpression is one of the alterations that characterizes the invasive phenotype and is considered an invasion/tumour suppressor gene. Partial or complete loss of E-Cadherin expression correlates with poor prognosis in canine malignant mammary cancer.The aim of this study was to analyse the sLex expression in canine malignant mammary tumours and to evaluate if the presence of sLex correlates with the expression of E-Cadherin and with clinicopathological features.MethodsFifty-three cases of canine mammary carcinomas were analysed immunohistochemically using monoclonal antibodies against sLex (IgM) and E-Cadherin (IgG). The clinicopathological data were then assessed to determine whether there was a correlation with sLex tumour expression. Double labelled immunofluorescence staining was performed to analyse the combined expression of sLex and E-Cadherin.ResultssLex expression was consistently demonstrated in all cases of canine mammary carcinomas with different levels of expression. We found a significant relationship between the levels of sLex expression and the presence of lymph node metastases. We also demonstrated that when E-Cadherin expression was increased sLex was reduced and vice-versa. The combined analysis of both adhesion molecules revealed an inverse relationship.ConclusionIn the present study we demonstrate the importance of sLex in the malignant phenotype of canine malignant mammary tumours. Our results support the use of sLex as a prognostic tumour marker in canine mammary carcinomas. Furthermore, we showed that sLex and E-Cadherin expression were inversely correlated. Future studies are warranted to clarify the molecular mechanism underlying the relation between sLex and E-Cadherin in canine mammary carcinoma cells which represents an important comparative model to woman breast cancer.