Rosa Peracaula

Altered Glycosylation in Tumours Focused to Cancer Diagnosis

The lack of specific and sensitive tumour markers for early detection of cancer is driving a search for new approaches that could identify biomarkers. Markers are needed to alert clinicians at the early stages of tumourogenesis, before the cancer has metastasized, when the therapeutic drugs are more effective. Most tumour markers currently used in clinics are serum glycoproteins, frequently highly glycosylated mucins. Typically, the disease marker is the protein and not the glycan moiety of the corresponding glycoprotein or mucin. The increasing knowledge of the role of glycans in cancer suggests that further studies may assist both in determining their role in every step of tumour progression, and in the design of new therapeutic and diagnosic approaches. Detection of the altered glycans in serum tumour glycoproteins could be a way to achieve specificity in tumour detection. In this review, we focus on the glycan changes of two serum glycoproteins, prostate specific antigen - currently used as a tumour marker of prostate cancer - and human pancreatic ribonuclease in pancreatic adenocarcinoma. The detection of glycan changes, associated with subsets of glycoforms in serum glycoproteins that are specific to the tumour situation, could be the basis for developing more specific biomarkers.

Glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis.

Purpose: Glycosylation of acute‐phase proteins (APP), which is partially regulated by cytokines, may be distinct in disease and provide useful tumour markers. Thus, we have examined the glycosylation of major serum APP in pancreatic cancer (PaC), chronic pancreatitis (CP) and control patients.

Glycan Characterization of PSA 2-DE Subforms from Serum and Seminal Plasma

Prostate-specific antigen (PSA) two-dimensional electrophoresis (2-DE) subforms (F1-F5) have been described to be altered in prostate cancer (PCa) compared to benign prostatic hyperplasia (BPH). To understand their molecular differences, characterization of these subforms from PCa serum and seminal plasma, namely, at the glycan level, was performed. PSA 2-DE subforms from two serum PCa samples and seminal plasma were analyzed by N-glycan sequencing using high-performance liquid chromatography (HPLC) combined with exoglycosidase array digestions and by mass spectrometry. F1, F2, and F3 subforms showed the same N-glycan pattern, which contained higher levels of sialic acid than the F4 subform, whereas the F5 subform was unglycosylated. When comparing PSA subforms from PCa with seminal plasma, a decrease in sialylation was observed. Furthermore, the analysis of F3, the more abundant PSA subform, showed a higher proportion of alpha 2-3 sialic acid and a decrease in core fucosylated glycans in the PCa sample. These N-glycan changes in PCa PSA subforms highlight the importance of glycosylation as an indicator of PCa disease.

5-AZA-2'-deoxycytidine induced demethylation influences N-glycosylation of secreted glycoproteins in ovarian cancer.

Glycosylation is the most common posttranslational modification of proteins and is highly reflective of changes in the environment of a cell. Epigenetic modifications to the genome are stably transmitted to daughter cells without the requirement for genetic sequence alterations. Aberrant regulation of both epigenetic programming and glycosylation patterning are integral aspects of carcinogenesis. The objective of this study was to determine the interplay between these two complex cellular processes. We demonstrate that global DNA methylation changes in ovarian cancer epithelial cells (OVCAR3) resulted in significant alterations in the glycosylation of secreted glycoproteins. These changes included a reduction in core fucosylation, increased branching and increased sialylation. We further show that the change in core fucose levels was mirrored by altered expression of GMDS and FX, key enzymes in fucose biosynthesis. Alterations in the expression of key glycosyltransferase enzymes such as MGAT5 reflect the changes seen in the branching and sialylation of secreted glycans. Overall, our results highlight that modifications to the epigenetic machinery have a profound effect on the glycan structures generated by cells, which may be a key step in understanding metastasis and drug resistance during cancer progression.

Differential percentage of serum prostate-specific antigen subforms suggests a new way to improve prostate cancer diagnosis.

Prostate‐specific antigen (PSA) is the tumor marker currently used for prostate cancer (PCa) screening and diagnosis. However, its use is controversial as serum PSA levels are also increased in other non‐malignant prostatic diseases such as benign prostatic hyperplasia (BPH). PSA sialic acid content is altered in tumor situation and modifies PSAs isoelectric point (pI). Our goal has been to evaluate serum PSA subforms from PCa and BPH patients by two‐dimensional electrophoresis (2‐DE) and to investigate whether they could be used to improve PCa diagnosis.

α2,3-Sialyltransferase ST3Gal IV promotes migration and metastasis in pancreatic adenocarcinoma cells and tends to be highly expressed in pancreatic adenocarcinoma tissues.

Sialyltransferases have received much attention recently as they are frequently up-regulated in cancer cells. However, the role played by each sialyltransferase in tumour progression is still unknown. α2,3-Sialyltransferases ST3Gal III and ST3Gal IV are involved in sialyl-Lewis(x) (SLe(x)) synthesis. Given that the role of ST3Gal III in pancreatic adenocarcinoma cells has been previously reported, in this study we have focused on investigating the role of ST3Gal IV in the acquisition of adhesive, migratory and metastatic capabilities and, secondly, in analyzing the expression of ST3Gal III and ST3Gal IV in pancreatic adenocarcinoma tissues versus control tissues. ST3Gal IV overexpressing pancreatic adenocarcinoma MDAPanc-28 cell lines were generated. They showed a heterogeneous increase in SLe(x), and enhanced E-selectin adhesion and migration. Furthermore, when injected into nude mice, increased metastasis and decreased survival were found in comparison with controls. The behaviour of MDAPanc-28 ST3Gal IV overexpressing cells in these processes was similar to the already reported MDAPanc-28 ST3Gal III overexpressing cells. Furthermore, pancreatic adenocarcinoma tissues tended to express high levels of ST3Gal III and ST3Gal IV together with other fucosyltransferase genes FUT3 and FUT6, all involved in the last steps of sialyl-Lewis(x) biosynthesis. In conclusion, both α2,3-sialyltransferases are involved in key steps of pancreatic tumour progression processes and are highly expressed in most pancreatic adenocarcinoma tissues.

Effect of sialic acid content on glycoprotein pI analyzed by two-dimensional electrophoresis

2‐DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2‐DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2‐DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2‐DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2‐DE pattern of three serum glycoproteins (haptoglobin, α1‐antitrypsin and ribonuclease 1), which N‐glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule.

Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes

New markers based on PSA isoforms have recently been developed to improve prostate cancer (PCa) diagnosis. However, novel approaches are still required to differentiate aggressive from non-aggressive PCa to improve decision making for patients. PSA glycoforms have been shown to be differentially expressed in PCa. In particular, changes in the extent of core fucosylation and sialylation of PSA N-glycans in PCa patients compared to healthy controls or BPH patients have been reported. The objective of this study was to determine these specific glycan structures in serum PSA to analyze their potential value as markers for discriminating between BPH and PCa of different aggressiveness. In the present work, we have established two methodologies to analyze the core fucosylation and the sialic acid linkage of PSA N-glycans in serum samples from BPH (29) and PCa (44) patients with different degrees of aggressiveness. We detected a significant decrease in the core fucose and an increase in the α2,3-sialic acid percentage of PSA in high-risk PCa that differentiated BPH and low-risk PCa from high-risk PCa patients. In particular, a cut-off value of 0.86 of the PSA core fucose ratio, could distinguish high-risk PCa patients from BPH with 90% sensitivity and 95% specificity, with an AUC of 0.94. In the case of the α2,3-sialic acid percentage of PSA, the cut-off value of 30% discriminated between high-risk PCa and the group of BPH, low-, and intermediate-risk PCa with a sensitivity and specificity of 85.7% and 95.5%, respectively, with an AUC of 0.97. The latter marker exhibited high performance in differentiating between aggressive and non-aggressive PCa and has the potential for translational application in the clinic.

Role of sialyltransferases involved in the biosynthesis of Lewis antigens in human pancreatic tumour cells

AbstractThe sialylated carbohydrate antigens, sialyl-Lewisx and sialyl-Lewisa, are expressed in pancreatic tumour cells and are related to their metastatic potential. While the action of the fucosyltransferases involved in the synthesis of these antigens has already been investigated, no studies have been carried out on the activity and expression of the α 2,3-sialyltransferases in pancreatic tumour cells. We describe the sialyltransferase (ST) activity, mRNA expression, and analysis of the cell carbohydrate structures in four human pancreatic adenocarcinoma cell lines of a wide range of neoplastic differentiation stages and in normal human pancreatic tissues. Total ST activity measured on asialofetuin, employing a CMP fluorescent sialic acid, varied among the pancreatic cell lines and could be correlated to the expression of their cell surface antigens. However, in some of the pancreatic cell lines, no relationship could be established with their ST3Gal III and IV mRNA expression. Human pancreatic tissues also showed ST expression and activity. However, it presented a much higher expression of neutral fucosylated structures than sialylated structures. In conclusion, ST activity levels in pancreatic cells could be correlated to their expression of sialylated epitopes, which indicates their involvement in the formation of the sialyl-Lewis antigens, in addition to fucosyltransferase activities. Published in 2005.

Liver proteins as sensor of human malignancies and inflammation.

In this review we would like to highlight the importance of acute‐phase proteins as sensor of diseases. Both acute‐phase protein levels and glycosylation have been reported to be altered in inflammation and other diseases including cancer. Factors that promote acute‐phase protein synthesis and enhance the expression of specific glycosyltransferases, such as sialyltransferases and fucosyltransferases, may be up‐regulated in some tumours and would explain the changes in acute‐phase protein levels and the specific N‐glycosylation modifications of some acute‐phase proteins in cancer. However, further studies are required to define the potential clinical application of these acute‐phase protein cancer‐specific modifications as possible cancer diagnostic or monitoring tools.