RESUMEN

Changes in the glycosylation process appear early in carcinogenesis and evolve with the growth and spread of cancer. The correlation of the characteristic glycosylation signature with the tumor stage and the appropriate therapy choice is an important issue in translational medicine. Oncologists also pay attention to extracellular vesicles as reservoirs of new cancer glycomarkers that can be potent for cancer diagnosis/prognosis. In this review, we recall glycomarkers used in oncology and show their new glycoforms of improved clinical relevance. We summarize current knowledge on the biological functions of glycoepitopes in cancer-derived extracellular vesicles and their potential use in clinical practice. Is glycomics a future of cancer diagnosis? It may be, but in combination with other omics analyses than alone.

Asunto(s)

Vesículas Extracelulares , Neoplasias , Humanos , Glicosilación , Neoplasias/diagnóstico , Neoplasias/terapia , Neoplasias/metabolismo , Carcinogénesis/metabolismo , Vesículas Extracelulares/metabolismo , Glicómica

RESUMEN

The algorithms commonly used to select the best stable reference gene in RT-qPCR data analysis have their limitations. We showed that simple selection of the reference gene or pair of genes with the lowest stability value from the pool of potential reference genes-a commonly used approach-is not sufficient to accurately and reliably normalize the target gene transcript and can lead to biologically incorrect conclusions. For reliable assessment of changes in a target gene expression level, we propose our innovative GenExpA software, which works in a manner independent of the experimental model and the normalizer used. GenExpA software selects the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and in the set of daughter models assigned to it. The reliability of references is validated based on the consistency of the statistical analyses of normalized target gene expression levels through all models, described by the coherence score (CS). The use of the CS value imparts a new quality to qPCR analysis because it clarifies how low the stability value of reference must be in order for biologically correct conclusions to be drawn. We tested our method on qPCR data for the B4GALT genes family in melanoma, which is characterized by a high mutation rate, and in melanocytes. GenExpA is available at https://github.com/DorotaHojaLukowicz/GenExpA or https://www.sciencemarket.pl/baza-programow-open-source#oferty .

Asunto(s)

Melanoma , Programas Informáticos , Expresión Génica , Perfilación de la Expresión Génica/métodos , Humanos , Melanoma/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de Referencia , Reproducibilidad de los Resultados

RESUMEN

Changes in glycosylation pattern of cell surface, body fluids and extracellular matrix glycoconjugates is a characteristic feature of tumor cell malignancy. These changes are the result of mutations of tumor-associated genes as well as epigenetic changes in the tumor environment, including nutrient influx, hypoxia, cytokine expression and stimulation of chronic inflammation. The unique set of cell surface glycoantigens on neoplastic cells is recognized by endogenous lectins located in the extracellular matrix, vascular endothelium, on leukocytes or platelets, and has an impact on disrupting basic cellular processes, such as intercellular recognition, cell-cell adhesion or cell-ECM interaction. These changes have a critical impact on the migration, invasive and metastatic potential of neoplastic cells and modulate the immune response. This unique pattern of sugar antigens on the cancer cells can be a vaulable marker to identify them, determine the stage of the disease as well as be a target of anti-cancer therapy.

Asunto(s)

Neoplasias , Adhesión Celular , Matriz Extracelular/metabolismo , Glicosilación , Humanos , Neoplasias/metabolismo , Polisacáridos/metabolismo

RESUMEN

Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC-MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial-mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.

Asunto(s)

Micropartículas Derivadas de Células/metabolismo , Exosomas/metabolismo , Melanoma/metabolismo , Proteómica , Neoplasias Cutáneas/metabolismo , Espectrometría de Masas en Tándem , Biomarcadores , Línea Celular Tumoral , Cromatografía Liquida , Biología Computacional/métodos , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Melanoma/genética , Neoplasias Cutáneas/genética , Melanoma Cutáneo Maligno

RESUMEN

Cancer cells are known to release extracellular vesicles that often promote disease development and progression. The present study investigated the protein content and glycosylation pattern of ectosomes released in vitro by a human primary uveal melanoma Mel202 cell line. Ectosomes released by Mel202 cells were isolated from conditioned media using sequential centrifugation, and a nano-LC-MS/MS approach was used to determine their protein content. Subsequently, proteins from ectosomes, the whole cell extracts, and the membrane fractions were probed with a panel of lectins using Western blotting and flow cytometry to reveal characteristic glycan structures. As many as 2527 unique proteins were identified, and many of them are known to be involved in cancer cell proliferation and altered metabolism, tumor invasion, metastasis, or drug resistance. Lectin-based studies revealed a distinct glycosylation pattern between Mel202-derived ectosomes and the parental cell membranes. Selective enrichment of ectosomal proteins with bisected complex type N-glycans and α2,6-linked sialic acids may be significant for ectosome formation and sequestration. Differences in the surface glycosylation of Mel202 cells and ectosomes supports recent findings that the budding of ectosomes occurs within strictly determined fragments of the plasma membrane, and thus ectosomes contain a unique protein and glycan composition.

Asunto(s)

Micropartículas Derivadas de Células/metabolismo , Melanoma/metabolismo , Proteoma/metabolismo , Neoplasias de la Úvea/metabolismo , Biomarcadores/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/patología , Micropartículas Derivadas de Células/patología , Glicosilación , Humanos , Melanoma/patología , Neoplasias de la Úvea/patología

RESUMEN

AIMS: Numerous studies confirmed the involvement of extracellular vesicles in cancer development and progression. The present study was designed to investigate the glycan composition of ectosomes derived by human cutaneous melanoma (CM) cell lines with the use of lectins. MAIN METHODS: Ectosomes released by primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cells were isolated from conditioned media by sequential centrifugation. Proteins from ectosomes, the whole cell extracts and membrane fractions were probed with a panel of lectins using Western Blot and flow cytometry and compared in terms of disease stage and glycosignature. KEY FINDINGS: Ectosomal proteins revealed enrichment (mainly with fucose and complex type N-glycans with bisecting GlcNAc) or depletion of specific glycoepitopes in comparison to the parental cell membranes. Moreover, similar lectin binding patterns were observed between related cell lines. It is the first study to characterize the glycosylation of ectosome proteins released by CM cells. SIGNIFICANCE: Our data indirectly supports the findings that ectosomes derive from particular regions of the cell membrane contain a unique glycan composition, which could serve as a specific sorting signal. If proven correct, the hypothesis that glycan-based protein sorting is a major mechanism for protein incorporation into ectosomes may provide new means to control vesicular content and have possible clinical implications.

Asunto(s)

Micropartículas Derivadas de Células/química , Epítopos/química , Melanoma/metabolismo , Polisacáridos/química , Neoplasias Cutáneas/metabolismo , Biomarcadores/química , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Progresión de la Enfermedad , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Glicosilación , Humanos , Lectinas/química , Metástasis Linfática , Melanoma Cutáneo Maligno

RESUMEN

The objective of this study was to identify a normalizer or combination of normalizers for quantitative evaluation of the expression of a target gene of interest during melanoma progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein polypeptide A (SRNPA) were chosen as candidate housekeeping genes. NormFinder software was used to identify the best reference gene or pair of reference genes from five candidate housekeeping genes, on the basis of expression stability in a given experimental model. The suitability of references was validated by normalizing the transcriptional activities of E-cadherin (CDH1), N-cadherin (CDH2) and endoplasmic reticulum aminopeptidase 1 (ERAP1) target genes. It has been shown that the relative expression of CDH2 and ERAP1 target genes in a given cell line may vary between experimental models, leading to biological misinterpretation. In view of this, we devised a strategy for improved selection of the best stable reference and for obtaining biologically consistent results. This strategy avoided experimental model- and normalizer-dependent conclusions concerning the relative expression of target gene, in the examined cell lines.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Genes Esenciales , Melanoma/genética , Proteínas de Neoplasias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Neoplasias de la Úvea/genética , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Glucuronidasa/genética , Glucuronidasa/metabolismo , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/metabolismo , Melanocitos/citología , Melanocitos/metabolismo , Melanoma/diagnóstico , Melanoma/metabolismo , Melanoma/patología , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoglicerato Quinasa/genética , Fosfoglicerato Quinasa/metabolismo , Cultivo Primario de Células , Estándares de Referencia , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Neoplasias de la Úvea/diagnóstico , Neoplasias de la Úvea/metabolismo , Neoplasias de la Úvea/patología

RESUMEN

Cutaneous melanoma, the most aggressive form of skin cancer, responds poorly to conventional therapy. The appearance of Tn antigen-modified proteins in cancer is correlated with metastasis and poor prognoses. The Tn determinant has been recognized as a powerful diagnostic and therapeutic target, and as an object for the development of anti-tumor vaccine strategies. This study was designed to identify Tn-carrying proteins and reveal their influence on cutaneous melanoma progression. We used a lectin-based strategy to purify Tn antigen-enriched cellular glycoproteome, the LC-MS/MS method to identify isolated glycoproteins, and the DAVID bioinformatics tool to classify the identified proteins. We identified 146 different Tn-bearing glycoproteins, 88% of which are new. The Tn-glycoproteome was generally enriched in proteins involved in the control of ribosome biogenesis, CDR-mediated mRNA stabilization, cell-cell adhesion and extracellular vesicle formation. The differential expression patterns of Tn-modified proteins for cutaneous primary and metastatic melanoma cells supported nonmetastatic and metastatic cell phenotypes, respectively. To our knowledge, this study is the first large-scale proteomic analysis of Tn-bearing proteins in human melanoma cells. The identified Tn-modified proteins are related to the biological and molecular nature of cutaneous melanoma and may be valuable biomarkers and therapeutic targets.

Asunto(s)

Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Biomarcadores de Tumor/metabolismo , Glicoproteínas/metabolismo , Melanoma/metabolismo , Proteómica , Neoplasias Cutáneas/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Humanos , Melanoma/patología , Metástasis de la Neoplasia , Neoplasias Cutáneas/patología , Espectrometría de Masas en Tándem

RESUMEN

Ectosomes are small heterogeneous membrane vesicles generated by budding from the plasma membrane in a variety of cell types and, more frequently, in tumor cells. They are shed into the extracellular space and are proposed as a novel form of intracellular communication in which information is transmitted from the originating cell to recipient cells without direct cell-to-cell contact. This review focuses on a single population of extracellular vesicles-ectosomes. We summarize recent studies of tumor-derived ectosomes which examine their biogenesis and protein cargo, and their influence on different aspects of cancer progression. We discuss possible clinical implications involving ectosomes as potential biomarkers, diagnostic tools and treatment targets in oncology. The unique composition of the molecules (cargo) that ectosomes carry, and their functional role, depends largely on the state of their originating cell. Through horizontal transfer of a variety of biologically active molecules (including proteins, lipids and nucleic acids) between donor and recipient cells, tumor-derived ectosomes may play functional roles in oncogenic transformation, tumor progression, invasion, metastasis, angiogenesis promotion, escape from immune surveillance, and drug resistance, thereby facilitating disease progression. The presence of tumor-derived ectosomes in body fluids such as the blood and urine of cancer patients makes them potentially useful prognostic and predictive biomarkers. Tumor-derived ectosomes also offer possible targets for multiple therapeutic strategies.

Asunto(s)

Transformación Celular Neoplásica/patología , Micropartículas Derivadas de Células/patología , Neoplasias/patología , Animales , Comunicación Celular , Progresión de la Enfermedad , Humanos

RESUMEN

Changes in the profile of protein glycosylation are a hallmark of ongoing neoplastic transformation. A unique set of tumor-associated carbohydrate antigens expressed on the surface of malignant cells may serve as powerful diagnostic and therapeutic targets. Cell-surface proteins with altered glycosylation affect the growth, proliferation and survival of those cells, and contribute to their acquisition of the ability to migrate and invade. They may also facilitate tumor-induced immunosuppression and the formation of distant metastases. Deciphering the information encoded in these particular glycan portions of glycoconjugates may shed light on the mechanisms of cancer progression and metastasis. A majority of the related review papers have focused on overall changes in the patterns of cell-surface glycans in various cancers, without pinpointing the molecular carriers of these glycan structures. The present review highlights the ways in which particular tumor-associated glycan(s) coupled with a given membrane-bound protein influence neoplastic cell behavior during the development and progression of cancer. We focus on altered glycosylated cell-adhesion molecules belonging to the cadherin, integrin and immunoglobulin-like superfamilies, examined in the context of molecular interactions.

Asunto(s)

Neoplasias/metabolismo , Neoplasias/patología , Polisacáridos/metabolismo , Animales , Adhesión Celular , Humanos , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Polisacáridos/química

RESUMEN

The present study investigated the influence of the androgen receptor (AR) agonists testosterone (T) and dihydrotestosterone (DHT), and vinclozolin (Vnz), a fungicide with antiandrogenic activity, on non-genomic signal transduction within ovarian follicles. Porcine granulosa cells (GCs) isolated from mature follicles were cultured for 48h. For the last 24h of culture, they were exposed to T (10(-7)M), DHT (10(-7)M), Vnz (1.4×10(-5)M), T and Vnz (T+Vnz), or DHT and Vnz (DHT+Vnz) at the same concentrations. To better imitate in vivo conditions, whole follicles (4-6mm in diameter) were incubated (24h) in an organ culture system with the same factors. Expression of AR mRNA and protein was determined by real-time PCR and western blot analyses. To demonstrate AR localization in cultured GCs and whole follicles, immunocytochemistry and immunohistochemistry were performed, respectively. To elucidate the possible non-genomic action of Vnz in GCs, protein expression and the activity of ERK1/2 and Akt kinases were determined by western blot and ELISA analyses. The immunocytochemistry and immunohistochemistry results showed that exposure of GCs and follicles to Vnz resulted in cytoplasmic and perinuclear AR localization. Real-time PCR and western blot analysis showed that AR mRNA and protein expression increased (P≤0.001) in GC cultures after combined treatment with an androgen and Vnz. In whole follicles, such treatment also increased AR mRNA with a decrease in the respective protein expression (P≤0.001). Moreover, addition of T or DHT with Vnz increased the activity of ERK1/2 and Akt kinases in cultured GCs (P≤0.001). The results suggest a novel mechanism for Vnz action in porcine ovarian follicles on both AR mRNA and protein levels. Thus, this environmental antiandrogen activates non-genomic signaling pathways, as indicated by the increased activity of both investigated kinases observed within minutes of Vnz addition. Given the widespread presence of Vnz in the environment, elucidation of its non-genomic action should be the subject of studies on female fertility.

Asunto(s)

Antagonistas de Andrógenos/farmacología , Células de la Granulosa/metabolismo , Oxazoles/farmacología , Receptores Androgénicos/metabolismo , Animales , Células Cultivadas , Dihidrotestosterona/farmacología , Evaluación Preclínica de Medicamentos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/genética , Transducción de Señal , Sus scrofa

RESUMEN

Melanoma is the most aggressive of all skin cancers and is exceptionally resistant to therapies. During melanoma progression, cancer cells reprogram their proliferation and survival pathways and achieve resistance to treatment-induced apoptosis. Galectin-3 (gal-3) is a member of the lectin family and is involved in such biological processes as cell adhesion, growth and differentiation, the cell cycle, and apoptosis. Gal-3 also plays an important role in tumor development and metastasis. The relationship between gal-3 expression and these processes is specific to the tumor type and the stage of cancer progression. The biological functions of gal-3 depend on its localization in the cell. In the present study, human metastatic melanoma A-375 cells, characterized by weak endogenous expression of gal-3, were transfected with gal-3 cDNA and cisplatin-induced apoptosis was measured. Data from AnnexinV and mitochondrial membrane potential analysis revealed that gal-3 did not protect the A-375 melanoma cells against cisplatin. This result probably is associated with its nuclear localization in the cells.

Asunto(s)

Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Galectina 3/metabolismo , Antineoplásicos/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Galectina 3/genética , Humanos , Melanoma/metabolismo , Melanoma/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección

RESUMEN

Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed.

Asunto(s)

Apoptosis/efectos de los fármacos , Fungicidas Industriales/toxicidad , Células de la Granulosa/efectos de los fármacos , Oxazoles/toxicidad , Porcinos/metabolismo , Animales , Apoptosis/fisiología , Células Cultivadas , Femenino , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Unlike nuclear nucleolin, surface-expressed and cytoplasmic nucleolin exhibit Tn antigen. Here, we show localization-dependent differences in the glycosylation and proteolysis patterns of nucleolin. Our results provide evidence for different paths of nucleolin proteolysis in the nucleus, in the cytoplasm, and on the cell surface. We found that full-length nucleolin and some proteolytic fragments coexist within live cells and are not solely the result of the preparation procedure. Extranuclear nucleolin undergoes N- and O-glycosylation, and unlike cytoplasmic nucleolin, membrane-associated nucleolin is not fucosylated. Here, we show for the first time that nucleolin and endogenous galectin-3 exist in the same complexes in the nucleolus, the cytoplasm, and on the cell surface of melanoma cells. Assessments of the interaction of nucleolin with galectin-3 revealed nucleolar co-localization in interphase, suggesting that galectin-3 may be involved in DNA organization and ribosome biogenesis.

Asunto(s)

Galectina 3/metabolismo , Lectinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Western Blotting , Fraccionamiento Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Glicosilación , Humanos , Espectrometría de Masas , Melanoma/metabolismo , Melanoma/patología , Microscopía Confocal , Unión Proteica , Proteolisis , Nucleolina

RESUMEN

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of the best-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) and its products, ß1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers. In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793 cell line) to the metastatic stage (WM1205Lu line), ß1,6 glycosylation of melanoma cell surface proteins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatment with swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was stronger in metastatic cells, probably due to the higher content of GlcNAc ß1,6-branched glycans on the main fibronectin receptors - integrins α5ß1 and α3ß1. Our results show that GlcNAc ß1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.

Asunto(s)

Acetilglucosamina/metabolismo , Fibronectinas/metabolismo , Integrina alfa3beta1/metabolismo , Integrina alfa5beta1/metabolismo , Melanoma/metabolismo , Polisacáridos/metabolismo , Movimiento Celular , Glicosilación , Humanos , Integrina alfa3beta1/genética , Integrina alfa5beta1/genética , Melanoma/patología , N-Acetilglucosaminiltransferasas/metabolismo , Metástasis de la Neoplasia

RESUMEN

Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose ß1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and ß1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galß1-4Galß1- epitope capped with sialic acid residues A1[3]G(4)2S2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.

Asunto(s)

Galactosa/química , Melanoma/química , Molécula L1 de Adhesión de Célula Nerviosa/química , Polisacáridos/química , Acetilglucosamina/química , Amino Azúcares/química , Biomarcadores de Tumor , Conformación de Carbohidratos , Secuencia de Carbohidratos , Línea Celular Tumoral , Movimiento Celular , Epítopos/química , Fucosa/química , Humanos , Melanoma/patología , Ácido N-Acetilneuramínico/química , Invasividad Neoplásica

RESUMEN

Nucleolin is multifunctional protein mainly present in nucleoli but also detected in cytoplasm and plasma membranes. Extranuclear nucleolin differs from the nuclear form by its glycosylation. Studies on expression of nucleolin in breast cancer suggest a possible association to the metastatic cascade. In the present study, Vicia villosa lectin (VVL) precipitation followed by subsequent polyacrylamide gel electrophoresis and mass spectrometry analysis demonstrates nucleolin as a VVL-positive glycoprotein expressed in melanoma. The presence of VVL-positive nucleolin in the melanoma cell membrane and cytoplasm was confirmed by confocal microscopy. Using bioinformatic peptide prediction programs, nucleolin was shown to contain multiple possible MHC class-I binding peptides in its sequence which makes nucleolin an interesting melanoma marker and target for immunodiagnostic and possibly therapeutic purposes.

Asunto(s)

Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Melanoma/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Neoplasias Cutáneas/metabolismo , Secuencia de Aminoácidos , Western Blotting , Membrana Celular/metabolismo , Nucléolo Celular/metabolismo , Biología Computacional , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Genes MHC Clase I , Glicoproteínas/metabolismo , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Espectrometría de Masas , Datos de Secuencia Molecular , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Células Tumorales Cultivadas , Nucleolina

RESUMEN

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.

Asunto(s)

Integrina alfa3beta1/química , Integrina alfaVbeta3/química , Melanoma/metabolismo , Oligosacáridos/química , Neoplasias Cutáneas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Humanos , Integrina alfa3beta1/aislamiento & purificación , Integrina alfa3beta1/metabolismo , Integrina alfaVbeta3/aislamiento & purificación , Integrina alfaVbeta3/metabolismo , Lectinas/metabolismo , Ganglios Linfáticos/patología , Melanoma/secundario , Metástasis de la Neoplasia , Oligosacáridos/aislamiento & purificación , Oligosacáridos/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Neoplasias Cutáneas/secundario , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vitronectina/metabolismo , Kalinina

RESUMEN

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary beta1-6-N-acetylglucosamine (beta1-6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing beta1-6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35--from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing beta1-6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing beta1-6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.

Asunto(s)

Melanoma/química , Línea Celular Tumoral , Humanos , Oligosacáridos de Cadena Ramificada/química , Fitohemaglutininas/química , Swainsonina/farmacología , Espectrometría de Masas en Tándem , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

The repertoire of oligosaccharide components of cellular glycoproteins significantly contributes to cell adhesion and communication. In tumor cells, alteration in cellular glycosylation may play a key role in giving rise to invasive and metastatic potential. Over 100 melanoma cell lines deposited in the ESTDAB Melanoma Cell Bank (Tubingen, Germany) were studied for the characteristic glycan composition related to tumor progression. Analysis of: (1) cell adhesion to extracellular matrix proteins--fibronectin, laminin, and collagen; (2) the expression of selected glycosyltransferases--alpha2,3(Gal beta1,3)- and alpha2,3(Gal beta1,4)-sialyltransferases, alpha1,2- and alpha1,3-fucosyltransferases, and N-acetylglucosaminyltransferase V; (3) characterization of N-glycans was carried out on uveal (4), primary cutaneous (6), and metastatic (96) melanoma cell lines. Results showed that uveal cells did not adhere to any of the substrates and, in general, possessed less glycans containing alpha-2,6- and alpha-2,3-linked sialic acid. The average number of polypeptides bearing beta-1,6-branched tri- and tetra antennary glycans (characteristic of the metastatic phenotype) were similar in uveal, primary cutaneous, and metastatic melanoma cell lines. Characterization of N-glycans may open a new perspective in the search for specific glycoproteins that could become targets for the therapeutic modulation of melanoma.

Asunto(s)

Adhesión Celular , Glicosiltransferasas/biosíntesis , Melanoma/patología , Polisacáridos/fisiología , Neoplasias Cutáneas/patología , Neoplasias de la Úvea/patología , Progresión de la Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Humanos , Metástasis de la Neoplasia/fisiopatología , Fenotipo , Polisacáridos/análisis , Células Tumorales Cultivadas