RESUMO
The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.
Assuntos
Glicoesfingolipídeos , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Gangliosídeos/metabolismo , Globosídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Humanos , Transdução de SinaisRESUMO
G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward ß-arrestin-1 and ß-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting ß-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling.
Assuntos
Prolina/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Sítio Alostérico , Motivos de Aminoácidos , Clonagem Molecular , Dimerização , Células HEK293 , Humanos , Cinética , Ligantes , Peptídeos/química , Engenharia de Proteínas/métodos , Transdução de Sinais , beta-Arrestinas/metabolismoRESUMO
The reversible transitions of cancer cells between epithelial and mesenchymal states comprise cellular and molecular processes essential for local tumor growth and respective dissemination. We report here that globoside glycosphingolipid (GSL) glycosyltransferase-encoding genes are elevated in epithelial cells and correlate with characteristic EMT signatures predictive of disease outcome. Depletion of globosides through CRISPR-Cas9-mediated deletion of the key enzyme A4GALT induces EMT, enhances chemoresistance, and increased CD24low/CD44high cells. The cholera toxin-induced mesenchymal-to-epithelial transition occurred only in cells with functional A4GALT. Cells undergoing EMT lost E-cadherin expression through epigenetic silencing at the promoter region of CDH1 However, in ΔA4GALT cells, demethylation was able to rescue E-cadherin-mediated cell-cell adhesion only in the presence of exogenous A4GALT. Overall, our data suggest another class of biomolecules vital for epithelial cancer cells and for maintaining cell integrity and function.Significance: This study highlights the essential role of glycosphingolipids in the maintenance of epithelial cancer cell properties. Cancer Res; 78(11); 2952-65. ©2018 AACR.
Assuntos
Transição Epitelial-Mesenquimal/genética , Galactosiltransferases/genética , Globosídeos/metabolismo , Glicoesfingolipídeos/genética , Animais , Antígeno CD24/genética , Sistemas CRISPR-Cas/genética , Caderinas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Epigênese Genética/genética , Células Epiteliais/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Receptores de Hialuronatos/genética , Regiões Promotoras Genéticas/genética , Peixe-ZebraRESUMO
The (neo-) lacto series glycosphingolipids (nsGSLs) comprise of glycan epitopes that are present as blood group antigens, act as primary receptors for human pathogens and are also increasingly associated with malignant diseases. Beta-1, 3-N-acetyl-glucosaminyl-transferase 5 (B3GNT5) is suggested as the key glycosyltransferase for the biosynthesis of nsGSLs. In this study, we investigated the impact of CRISPR-Cas9 -mediated gene disruption of B3GNT5 (∆B3GNT5) on the expression of glycosphingolipids and N-glycoproteins by utilizing immunostaining and glycomics-based PGC-UHPLC-ESI-QTOF-MS/MS profiling. ∆B3GNT5 cells lost nsGSL expression coinciding with reduction of α2-6 sialylation on N-glycoproteins. In contrast, disruption of B4GALNT1, a glycosyltransferase for ganglio series GSLs did not affect α2-6 sialylation on N-glycoproteins. We further profiled all known α2-6 sialyltransferase-encoding genes and showed that the loss of α2-6 sialylation is due to silencing of ST6GAL1 expression in ∆B3GNT5 cells. These results demonstrate that nsGSLs are part of a complex network affecting N-glycosylation in ovarian cancer cells.
Assuntos
Glicoproteínas/metabolismo , Glicoesfingolipídeos/metabolismo , N-Acetilgalactosaminiltransferases/genética , Neoplasias Ovarianas/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Glicômica , Células HeLa , Humanos , Neoplasias Ovarianas/genéticaRESUMO
BACKGROUND: Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. RESULTS: We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. CONCLUSIONS: Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.
Assuntos
Anticorpos/sangue , Anticorpos/imunologia , Antígenos Glicosídicos Associados a Tumores/imunologia , Glucanos/imunologia , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/imunologia , Anticorpos/metabolismo , Biomarcadores Tumorais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/sangue , Cistadenocarcinoma Seroso/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Ovarianas/diagnóstico , Ligação Proteica/imunologiaRESUMO
Glucosylceramide synthase (GCS) catalyzes the first committed step in the biosynthesis of glucosylceramide (GlcCer)-related glycosphingolipids (GSLs). Although inhibitors of GCS, PPMP and PDMP have been widely used to elucidate their biological function and relevance, our comprehensive literature review revealed that the available data are ambiguous. We therefore investigated whether and to what extent GCS inhibitors affect the expression of lactosylceramide (LacCer), neolacto (nLc4 and P1), ganglio (GM1 and GD3) and globo (Gb3 and SSEA3) series GSLs in a panel of human cancer cell lines using flow cytometry, a commonly applied method investigating cell-surface GSLs after GCS inhibition. Their cell-surface GSL expression considerably varied among cell lines and more importantly, sublethal concentrations (IC10) of both inhibitors preferentially and significantly reduced the expression of Gb3 in the cancer cell lines IGROV1, BG1, HT29 and T47D, whereas SSEA3 was only reduced in BG1. Unexpectedly, the neolacto and ganglio series was not affected. LacCer, the precursor of all GlcCer-related GSL, was significantly reduced only in BG1 cells treated with PPMP. Future research questions addressing particular GSLs require careful consideration; our results indicate that the extent to which there is a decrease in the expression of one or more particular GSLs is dependent on the cell line under investigation, the type of GCS inhibitor and exposure duration.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Glicoesfingolipídeos/biossíntese , Meperidina/análogos & derivados , Morfolinas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glucosiltransferases/metabolismo , Humanos , Meperidina/farmacologiaRESUMO
A newly synthesized Nickel (II) tyrosine complex was screened as potential antimicrobial agent against a number of medically important bacteria (Bacillus subtilis, Streptococcus ß-haemolytica, Escherichia coli, Shigella dysenterae) and fungi (Aspergillus fumigatus, Candida albicans, Aspergillus niger, Aspergillus flavus, Penicillium sp.) strains. were used for antifungal activity. The antimicrobial activity was evaluated using the Agar Disc method. Moreover, the minimum inhibitory concentration of the complexes was determined against the same pathogenic bacteria and the values were found between 4~64 µg ml (-1). Brine shrimp bioassay was carried out for cytotoxicity measurements of the complexes. The LC50 values were calculated after probit transformation of the resulting mortality data and found to be 6 µg ml (-1).