A carregar...
Projeto de investigação
Exploiting Glycosylation of Colorectal Cancer for the development of improved diagnostics and therapeutics
Financiador
Autores
Publicações
Staining of E-selectin ligands on paraffin-embedded sections of tumor tissue
Publication . Carrascal, Mylène A.; Talina, Catarina; Borralho, Paula; Mineiro, A. Gonçalo; Henriques, Ana Raquel; Pen, Cláudia; Martins, Manuela; Braga, Sofia; Sackstein, Robert; Videira, Paula A.; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Centro de Estudos de Doenças Crónicas (CEDOC); UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; BioMed Central (BMC)
Background: The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLeX and sLeA), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLeX and/or sLeA. However, antibody binding does not define E-selectin binding activity. Methods: In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. Results: E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLeX/A, the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. Conclusions: The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis.
L1cam as an e-selectin ligand in colon cancer
Publication . Deschepper, Fanny M.; Zoppi, Roberta; Pirro, Martina; Hensbergen, Paul J.; Dall’olio, Fabio; Kotsias, Maximillianos; Gardner, Richard A.; Spencer, Daniel I. R.; Videira, Paula A.; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; MDPI - Multidisciplinary Digital Publishing Institute
Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.
MHC class I stability is modulated by cell surface sialylation in human dendritic cells
Publication . Silva, Zélia; Ferro, Tiago; Almeida, Danielle; Soares, Helena; Ferreira, José Alexandre; Deschepper, Fanny M.; Hensbergen, Paul J.; Pirro, Martina; van Vliet, Sandra J.; Springer, Sebastian; Videira, Paula A.; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Centro de Estudos de Doenças Crónicas (CEDOC); MDPI AG
Maturation of human Dendritic Cells (DCs) is characterized by increased expression of antigen presentation molecules, and overall decreased levels of sialic acid at cell surface. Here, we aimed to identify sialylated proteins at DC surface and comprehend their role and modulation. Mass spectrometry analysis of DC’s proteins, pulled down by a sialic acid binding lectin, identified molecules of the major human histocompatibility complex class I (MHC-I), known as human leucocyte antigen (HLA). After desialylation, DCs showed significantly higher reactivity with antibodies specific for properly folded MHC-I-β2-microglobulin complex and for β2-microglobulin but showed significant lower reactivity with an antibody specific for free MHC-I heavy chain. Similar results for antibody reactivities were observed for TAP2-deficient lymphoblastoid T2 cells, which express HLA-A*02:01. Using fluorescent peptide specifically fitting the groove of HLA-A*02:01, instead of antibody staining, also showed higher peptide binding on desialylated cells, confirming higher surface expression of MHC-I complex. A decay assay showed that desialylation doubled the half-life of MHC-I molecules at cell surface in both DCs and T2 cells. The biological impact of DC´s desialylation was evaluated in co-cultures with autologous T cells, showing higher number and earlier immunological synapses, and consequent significantly increased production of IFN-γ by T cells. In summary, sialic acid content modulates the expression and stability of complex MHC-I, which may account for the improved DC-T synapses.
Sialyl LewisX/A and Cytokeratin Crosstalk in Triple Negative Breast Cancer
Publication . Pascoal, Carlota; Carrascal, Mylène A.; Barreira, Daniela F.; Lourenço, Rita A.; Granjo, Pedro; Grosso, Ana R.; Borralho, Paula; Braga, Sofia; Videira, Paula A.; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); MDPI - Multidisciplinary Digital Publishing Institute
Triple-negative breast cancer (TNBC) encompasses multiple entities and is generally highly aggressive and metastatic. We aimed to determine the clinical and biological relevance of Sialyl-Lewis X and A (sLeX/A)—a fucosylated glycan involved in metastasis—in TNBC. Here, we studied tissues from 50 TNBC patients, transcripts from a TNBC dataset from The Cancer Genome Atlas (TCGA) database, and a primary breast cancer cell line. All 50 TNBC tissue samples analysed expressed sLeX/A. Patients with high expression of sLeX/A had 3 years less disease-free survival than patients with lower expression. In tissue, sLeX/A negatively correlated with cytokeratins 5/6 (CK5/6, which was corroborated by the inverse correlation between fucosyltransferases and CK5/6 genes. Our observations were confirmed in vitro when inhibition of sLeX/A remarkably increased expression of CK5/6, followed by a decreased proliferation and invasion capacity. Among the reported glycoproteins bearing sLeX/A and based on the STRING tool, α6 integrin showed the highest interaction score with CK5/6. This is the first report on the sLeX/A expression in TNBC, highlighting its association with lower disease-free survival and its inverse crosstalk with CK5/6 with α6 integrin as a mediator. All in all, sLeX/A is critical for TNBC malignancy and a potential prognosis biomarker and therapeutic target.
Unidades organizacionais
Descrição
Palavras-chave
Contribuidores
Financiadores
Entidade financiadora
European Commission
Programa de financiamento
H2020
Número da atribuição
676421