Inflammatory cytokines regulate the expression of glycosyltransferases involved in the biosynthesis of tumor-associated sialylated glycans in pancreatic cancer cell lines.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma containing several pro-inflammatory cytokines, which are described to modulate the expression of important genes related to tumor promotion and progression. In the present work we have investigated the potential role of these cytokines in the biosynthesis of tumor-associated carbohydrate antigens such as sialyl-Lewis(x) (SLe(x)) through the regulation of specific glycosyltransferase genes. METHODS: Two human PDAC cell lines MDAPanc-3 and MDAPanc-28 were treated with pro-inflammatory cytokines IL-1ß, TNFα, IL-6 or IL-8, and the content of tumor-associated carbohydrate antigens at the cell membrane was analyzed by flow cytometry. In addition, variation in the mRNA expression of sialyltransferase (ST) and fucosyltransferase (FUT) genes, which codify for the ST and FucT enzymes involved in the carbohydrate antigens' biosynthesis, was determined. The inflammatory microenvironment of PDAC tissues and the expression of Lewis-type antigens were analyzed by immunohistochemistry to find a possible correlation between inflammation status and the presence of tumor-associated carbohydrate antigens. RESULTS: IL-1ß stimuli increased SLe(x) and α2,6-sialic acid levels in MDAPanc-28 cells and enhanced the mRNA levels of ST3GAL3-4 and FUT5-7, which codify for ST and FucT enzymes related to SLe(x) biosynthesis, and of ST6GAL1. IL-6 and TNFα treatments increased the levels of SLe(x) and Le(y) antigens in MDPanc-3 cells and, similarly, the mRNA expression of ST3GAL3-4, FUT1-2 and FUT6, related to these Lewis-type antigens' biosynthesis, were increased. Most PDAC tissues stained for SLe(x) and SLe(a) and tended to be expressed in the tumor samples with a higher presence of inflammatory immune cells. CONCLUSIONS: The inflammatory microenvironment can modulate the glycosylation pattern of PDAC cells, increasing the expression of tumor-associated sialylated antigens such as SLe(x), which contributes to pancreatic tumor malignancy.

Pancreatic cancer cell glycosylation regulates cell adhesion and invasion through the modulation of α2ß1 integrin and E-cadherin function.

In our previous studies we have described that ST3Gal III transfected pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show increased membrane expression levels of sialyl-Lewis x (SLe(x)) along with a concomitant decrease in α2,6-sialic acid compared to control cells. Here we have addressed the role of this glycosylation pattern in the functional properties of two glycoproteins involved in the processes of cancer cell invasion and migration, α2ß1 integrin, the main receptor for type 1 collagen, and E-cadherin, responsible for cell-cell contacts and whose deregulation determines cell invasive capabilities. Our results demonstrate that ST3Gal III transfectants showed reduced cell-cell aggregation and increased invasive capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLe(x) and lower α2,6-sialic acid content on the glycans of their α2ß1 integrin molecules. As a consequence, higher phosphorylation of focal adhesion kinase tyrosine 397, which is recognized as one of the first steps of integrin-derived signaling pathways, was observed in these cells upon adhesion to type 1 collagen. This molecular mechanism underlies the increased migration through collagen of these cells. In addition, the pancreatic adenocarcinoma cell lines as well as human pancreatic tumor tissues showed colocalization of SLe(x) and E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion, changes in the sialylation pattern of α2ß1 integrin and E-cadherin appear to influence the functional role of these two glycoproteins supporting the role of these glycans as an underlying mechanism regulating pancreatic cancer cell adhesion and invasion.

Cell surface sialic acid modulates extracellular matrix adhesion and migration in pancreatic adenocarcinoma cells.

OBJECTIVES: Tumor cells modulate their extracellular matrix (ECM) adhesion and migration to become more metastatic. Moreover, they show an increase in sialic acid, which could have an effect on their ECM adhesion and migration. This work describes the influence of pancreatic adenocarcinoma cell surface α2,3- and α2,6-sialic acid determinants on the aforementioned processes. METHODS: We have characterized the cell surface α2,3- and α2,6-sialic acids, and sialyl-Lewis x levels and the integrin levels of 2 pancreatic adenocarcinoma cell lines, Capan-1 and MDAPanc-28, grown at different cell densities, and also of the ST3Gal III overexpressing Capan-1 cells, C31. We have measured their adhesion to several ECM proteins and their migration through collagen with and without blocking their sialic acid determinants. RESULTS: Adhesion to ECM proteins of Capan-1 and MDAPanc-28 grown at different cell densities, and of C31, depended on their cell surface sialic acid determinants repertoire, correlating the higher α2,6-sialic acid levels with their increased ECM adhesion. Cell migration also depended on their sialic acid determinants expression; and in this case, higher α2,3-sialic acid levels correlated with a more migratory phenotype. CONCLUSION: This study shows how the intrinsic heterogeneity of cell membrane sialylation regulates the adhesive and migratory potential of pancreatic adenocarcinoma cells.