Glycan Signature Changes in Stem Cell Differentiation and Cancer
Patil, Shilpa A.
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Glycosyltransferase (glycoT) enzymes mediate the transfer of monosaccharide from nucleotide-sugars to carbohydrate, lipid and protein based acceptors leading to formation of glycans. The regulation of the glycan structures thus formed occurs at 3 different levels viz. gene expression, protein/enzyme expression and structure expression on cells. We reviewed these different levels of regulation in cancer system. Further, these enzymes are developmentally regulated. So, we have also reviewed the glycosylation process in the stem cell system. Next, the interface between the cancer system and stem cells is the cancer stem cells (CSC). Glycan based structures are gaining importance in CSC biology where glycan biomarkers are pursued to isolate and purify CSCs for further investigations. We have reviewed such CSC glycans and their functional relevance known so far. The review of glycosylation in cancer and stem cells brought out the missing links during the regulation process. The connections between gene expression and enzyme expression, and enzyme expression and glycan expression are not very clear in most instances. In this work, we sought to draw the connections for some systems. Towards this end we employed RP-TLC technique to assay the enzyme expression in small volumes, using three customized methods. The small volume approaches we developed are versatile and can be easily used without having to deal with complex chemistry or immobilization techniques, which is a norm in array based assays. Our methods utilize a range of natural and synthetic acceptors for the analysis of complex biological specimen that have limited availability. We applied this strategy to analyze the glycosyltransferase expression in mouse embryonic stem cell (mESC) systems. We saw specific enzyme regulation as the mouse embryonic stem cells differentiated towards cardiomyocytes. These studies demonstrate an increase in overall β(1,3)galactosyltransferase and α(2,3)sialyltransferase activity, and a decrease in α(1,3)fucosyltransferases when these cells differentiate towards cardiomyocytes. Enzymatic and lectin binding data suggest a transition from Le X type structures in mESCs to sialylated Galβ1,3GalNAc type glycans upon differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated to cardiomyocytes. Thus, we developed simple, rapid, quantitative and scalable glycoT activity analysis methods and brought out the link between enzyme expression and structure in mESC system. Similarly, we sought to analyze this connection in cancer system using the methodology we developed here. Aberrant cell-surface glycosylation is associated with cancer. Accumulating evidence suggests that such glycan changes may be driven by alteration in the expression of Golgi localized glycoTs. Since terminal glycan modifications regulate cancer cell signaling/adhesion, and such determinants may serve as disease biomarkers, we evaluated the activity of sialyltransferases (sialylTs), fucosyltransferases (fucTs) and galactosyltransferases (galTs) acting on Galβ1,3GlcNAc (Type I), (Galβ1,4GlcNAc) Type II, and Galβ1,3GalNAc (Type III) substrates. Results using a panel of breast cancer cell lines (ZR-75-1, MCF7, T-47D, DU4475) demonstrate elevated α2,3/6sialylT activity towards Type III substrates, elevated α1,3/4fucT activity on Type I and II glycans, and enhanced β1,3galT function that can lead to Type III/Galβ1,3GalNAc epitopes. Additionally, tumor cells in primary human breast and colon tissue, as opposed to adjacent normal controls, expressed elevated β1,3galT and α2,3sialylT activity that can form the α(2,3)sialylated Type III glycans. Prostate cancer tissue did not exhibit such elevated enzyme activities. α1,3/4fucT activity was also higher in breast, but not colon or prostate tissue. The enzymology based prediction of enhanced α(2,3)sialylated Type III structures in breast and colon tissue was verified by performing histochemical analysis of tissue sections and microarrays (TMAs) of breast and colon cancer. No staining was seen in tumor without sialidase treatment. Also, the staining after sialidase treatment was high in primary tumors in comparison to the metastatic samples. It also correlated negatively with the HER-2 status. Thus, it indicates a negative correlation between the staining pattern and aggressiveness of the disease. Together, we developed a scheme to identify unique glycan structures or signatures in different cell systems. In this work, we demonstrated a direct correlation between enzyme activity and structural expression of glycans in embryonic stem cells and cancer system.