Dissecting the molecular function of neutral glycosphingolipids in ovarian cancer progression

Glycosphingolipids (GSLs) are essential structural components of each eukaryotic cell and comprise a heterogeneous group of membrane lipids covalently linked to a glycan moiety. GSLs play fundamental role in proliferation, differentiation, adhesion, protein and lipid trafficking, signalling events and binding ligands of bacterial toxin and viruses. Aberrant glycosylation on GSLs has been associated with malignant transformation. The presence of tumor associated GSLs has also been observed in epithelial ovarian cancer, which is the eight most common cause of cancer in the women worldwide bearing the highest mortality rate among all gynaecological cancers. Several studies from our group suggest a potential role of GSLs (Gb3 and P1) in ovarian cancer. Both are the members of neutral GSLs belonging to globo series (Gb3) and (neo-) lacto series (P1). The α1-4 galactosyltransferase (A4GALT) terminates both Gb3 and P1 in the GSLs biosynthesis pathway. Gb3 is expressed on different human cell types (erythrocytes, monocytes, granulocytes, fibroblasts, smooth muscle cells of digestive tract and urogenital system, and various cancer cell lines) as well as in cancer tissues (primary lesions of metastatic colon cancer and the colon cancer metastases to liver) while the presence of P1 is only known to be on erythrocytes. The functional role of these neutral GSLs has not been well understood in general and particular in context of ovarian cancer. To address this question, here we utilized two strategies: 1) enzymatic inhibition of key glycosyltransferase glucosylceramide synthase (GCS) and 2) CRISPR-Cas9 mediated genome editing to generate homozygous, stable, and heritable knockout cell lines to consequently establish an experimental tool depleted for specific glycan’s. The effects of inhibition and abolishment of these GSLs on cellular process is investigated with the appropriate assays. Our results displayed that ablation of (neo-) lacto series GSLs by CRISPR-Cas9 mediated genome editing (ΔB3GNT5) leads an unexpected loss of α 2-6 sialylation on N-glycoproteins. Profiling of α 2-6 sialyltransferase encoding genes revealed that loss of α 2-6 sialylation is due to the silencing of ST6GAL1 expression. Another study reported that globoside glycosyltransferases are elevated in epithelial signature and depletion of globosides by CRISPR-Cas9 mediated A4GALT deletion induce epithelial to mesenchymal transition (EMT) and consequently enhance chemo resistances. Cells undergoing EMT lost E-cadherin expression through epigenetic silencing at the promoter of CDH1 via DNA methylation, however, in A4GALT deleted cells demethylation was only able to rescue E-cadherin expression while wild type A4GALT was provided. Our data demonstrated another class of biomolecules vital for epithelial homeostasis to maintaining cell integrity and function. Taken together, our studies demonstrate that GSLs, and in particular globosides, play an important role in the transition of ovarian cancer cells towards epithelial (MET) or mesenchymal (EMT) phenotypes. Thus, these data suggest that particular GSLs are involved in a complex network affecting molecular events such as alteration of α 2,6- sialylation on N-glycoproteins and influencing E-cadherin mediated cell-cell adhesion in intermediate EMT ovarian cancer cell lines. In summary, this PhD thesis highlights a close relationship between the EMT process and GSLs, which allows a new direction for targeting new therapies.