Differential glycosylation of MUC1 and CEACAM5 between normal mucosa and tumour tissue of colon cancer patients.

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C-type lectins MGL and DC-SIGN, and Galectin-3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen-Friedenreich antigen (TF-antigen) (Core 1, Galß1-3GalNAc-Ser/Thr). Only small amounts of Tn-antigen (GalNAcα-Ser/Thr) expression was observed, but the human C-type lectin MGL showed increased binding to tumour-associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour-associated CEA (CEACAM5) contained high levels of the blood-group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C-type lectin DC-SIGN to tumour-associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N-glycans were prominent, and this correlated well with human Galectin-3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease.

Differential O- and Glycosphingolipid Glycosylation in Human Pancreatic Adenocarcinoma Cells With Opposite Morphology and Metastatic Behavior.

Changes in the glycosylation profile of cancer cells have been strongly associated with cancer progression. To increase our insights into the role of glycosylation in human pancreatic ductal adenocarcinoma (PDAC), we performed a study on O-glycans and glycosphingolipid (GSL) glycans of the PDAC cell lines Pa-Tu-8988T (PaTu-T) and Pa-Tu-8988S (PaTu-S). These cell lines are derived from the same patient, but show an almost opposite phenotype, morphology and capacity to metastasize, and may thus provide an attractive model to study the role of glycosylation in progression of PDAC. Gene-array analysis revealed that 24% of the glycosylation-related genes showed a ≥ 1.5-fold difference in expression level between the two cell lines. Subsequent validation of the data by porous graphitized carbon nano-liquid chromatography coupled to a tandem ion trap mass spectrometry and flow cytometry established major differences in O-glycans and GSL-glycans between the cell lines, including lower levels of T and sialylated Tn (sTn) antigens, neoexpression of globosides (Gb3 and Gb4), and higher levels of gangliosides in the mesenchymal-like PaTu-T cells compared to the epithelial-like PaTu-S. In addition, PaTu-S cells demonstrated a significantly higher binding of the immune-lectins macrophage galactose-type lectin and galectin-4 compared to PaTu-T. In summary, our data provide a comprehensive and differential glycan profile of two PDAC cell lines with disparate phenotypes and metastatic behavior. This will allow approaches to modulate and monitor the glycosylation of these PDAC cell lines, which opens up avenues to study the biology and metastatic behavior of PDAC.

Profiling of different pancreatic cancer cells used as models for metastatic behaviour shows large variation in their N-glycosylation.

To characterise pancreatic cancer cells from different sources which are used as model systems to study the metastatic behaviour in pancreatic ductal adenocarcinoma (PDAC), we compared the N-glycan imprint of four PDAC cells which were previously shown to differ in their galectin-4 expression and metastatic potential in vivo. Next to the sister cell lines Pa-Tu-8988S and Pa-Tu-8988T, which were isolated from the same liver metastasis of a PDAC, this included two primary PDAC cell cultures, PDAC1 and PDAC2. Additionally, we extended the N-glycan profiling to a normal, immortalized pancreatic duct cell line. Our results revealed major differences in the N-glycosylation of the different PDAC cells as well as compared to the control cell line, suggesting changes of the N-glycosylation in PDAC. The N-glycan profiles of the PDAC cells, however, differed vastly as well and demonstrate the diversity of PDAC model systems, which ultimately affects the interpretation of functional studies. The results from this study form the basis for further biological evaluation of the role of protein glycosylation in PDAC and highlight that conclusions from one cell line cannot be generalised, but should be regarded in the context of the corresponding phenotype.

High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells.

The current protocols for glycomic analysis of cells often require a large quantity of material (5-20 million cells). In order to analyze the N-glycosylation from small amounts of cells (≤1 million) as obtained from, for example, primary cell lines or cell sorting, and in a higher throughput approach, we set up a robust 96-well format PVDF-membrane based N-glycan release protocol followed by linkage-specific sialic acid stabilization, cleanup, and MALDI-TOF-MS analysis. We further evaluated the influence of PNGase F incubation time on the N-glycan profile.

Transcription elongation factor Spt4 mediates loss of phosphorylated RNA polymerase II transcription in response to DNA damage.

Previously, we found that Rad26, the yeast Cockayne syndrome B homolog and the transcription elongation factor Spt4 mediate transcription-coupled repair of UV-induced DNA damage. Here we studied the effect of DNA damage on transcription by directly analyzing the RNA polymerase II localization at active genes in vivo. A rad26 defect leads to loss of Ser5 phosphorylated RNA polymerase II localization to active genes, while localization is only transiently diminished in wild type cells. In contrast, loss of Ser5-P RNAP II localization is suppressed in spt4 cells. Interestingly, even when DNA damage is persistent the absence of Spt4 leads to a delayed loss of transcription suggesting that Spt4 is directly involved in mediating transcription shutdown. Comparative analysis of phosphorylated and non-phosphorylated RNA polymerase II localization revealed that Ser5-P RNAP II is preferentially lost in the presence of DNA damage. In addition, we found evidence for a transient Rad26 localization to active genes in response to DNA damage. These findings provide insight into the transcriptional response to DNA damage and the factors involved in communicating this response, which has direct implications for our understanding of transcription-repair coupling.

Hypoxia inducible factor 1α down regulates cell surface expression of α1,2-fucosylated glycans in human pancreatic adenocarcinoma cells.

The α1,2-fucosyltransferase activity in pancreatic tumors is much lower compared to normal pancreatic tissue. Here we show that hypoxia inducible factor (HIF) 1α is constitutively expressed in the pancreatic cancer cell lines Pa-Tu-8988S and Pa-Tu-8988T and suppresses the expression of the α1,2-fucosyltransferase genes FUT1 and FUT2. Down regulation of HIF-1α expression resulted in elevated FUT1 and FUT2 transcript levels and an increased expression of α1,2-fucosylated glycan structures on the surface of these cells. In conclusion, our data are the first to identify HIF-1α as a suppressor of FUT1/2 expression, thereby regulating α1,2-fucosylation of cell-surface glycans.

Galectin-4 expression is associated with reduced lymph node metastasis and modulation of Wnt/ß-catenin signalling in pancreatic adenocarcinoma.

Galectin-4 (Gal-4) has been recently identified as a pivotal factor in the migratory capabilities of a set of defined pancreatic ductal adenocarcinoma (PDAC) cell lines using zebrafish as a model system. Here we evaluated the expression of Gal-4 in PDAC tissues selected according to their lymph node metastatic status (N0 vs. N1), and investigated the therapeutic potential of targeting the cross-link with the Wnt signaling pathway in primary PDAC cells. Analysis of Gal-4 expression in PDACs showed high expression of Gal-4 in 80% of patients without lymph node metastasis, whereas 70% of patients with lymph node metastases had low Gal-4 expression. Accordingly, in primary PDAC cells high Gal-4 expression was negatively associated with migratory and invasive ability in vitro and in vivo. Knockdown of Gal-4 in primary PDAC cells with high Gal-4 expression resulted in significant increase of invasion (40%) and migration (50%, P<0.05), whereas enforced expression of Gal-4 in primary cells with low Gal-4 expression reduced the migratory and invasive behavior compared to the control cells. Gal-4 markedly reduces ß-catenin levels in the cell, counteracting the function of Wnt signaling, as was assessed by down-regulation of survivin and cyclin D1. Furthermore, Gal-4 sensitizes PDAC cells to the Wnt inhibitor ICG-001, which interferes with the interaction between CREB binding protein (CBP) and ß-catenin. Collectively, our data suggest that Gal-4 lowers the levels of cytoplasmic ß-catenin, which may lead to lowered availability of nuclear ß-catenin, and consequently diminished levels of nuclear CBP-ß-catenin complex and reduced activation of the Wnt target genes. Our findings provide novel insights into the role of Gal-4 in PDAC migration and invasion, and support the analysis of Gal-4 for rational targeting of Wnt/ß-catenin signaling in the treatment of PDAC.

Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells.

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.