Abstract
BackgroundGlycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC). Emerging evidences indicate significant involvement of cancer stem cells (CSCs) in PC aggressiveness. However, the importance of glycosylation in pancreatic cancer stem cells (PCSCs) is yet to be addressed. Hence, we evaluated the potential role of glycosylation in maintenance of stemness of PCSCs.MethodsEffect of glycosylation specific inhibitors on growth and PCSCs of PC cells was assessed by MTT assay and Side Population (SP) analysis. Isolated PCSCs/SP were characterized using molecular and functional assays. Expression of tumor-associated carbohydrate antigens (TACAs) was analyzed in PCSCs by western blotting. Effect of tunicamycin on PCSCs was analyzed by tumorsphere, clonogenicity, migration assay and immunoblotting for CSCs markers. The differential expression of glycogenes in PCSCs compared to non-CSCs were determined by RT-qPCR, immunoblotting and immunofluorescence. Co-expression of GALNT3 and B3GNT3 with CD44v6 was assessed in progression stages of KrasG12D; Pdx-1-Cre (KC) and KrasG12D; p53R172H; Pdx-1-Cre (KPC) tumors by immunofluorescence. Transient and CRISPR/Cas9 silencing of GALNT3 and B3GNT3 was performed to examine their effect on CSCs maintenance.ResultsInhibition of glycosylation decreased growth and CSCs/SP in PC cells. PCSCs overexpressed CSC markers (CD44v6, ESA, SOX2, SOX9 and ABCG2), exhibited global expressional variation of TACAs and showed higher self-renewal potential. Specifically, N-glycosylation inhibition, significantly decreased tumorsphere formation, migration, and clonogenicity of PCSCs, as well as hypo-glycosylated CD44v6 and ESA. Of note, glycosyltransferases (GFs), GALNT3 and B3GNT3, were significantly overexpressed in PCSCs and co-expressed with CD44v6 at advanced PDAC stages in KC and KPC tumors. Further, GALNT3 and B3GNT3 knockdown led to a decrease in the expression of cell surface markers (CD44v6 and ESA) and self-renewal markers (SOX2 and OCT3/4) in PCSCs. Interestingly, CD44v6 was modified with sialyl Lewis a in PCSCs. Finally, CRISPR/Cas9-mediated GALNT3 KO significantly decreased self-renewal, clonogenicity, and migratory capacity in PCSCs.ConclusionsTaken together, for the first time, our study showed the importance of glycosylation in mediating growth, stemness, and maintenance of PCSCs. These results indicate that elevated GALNT3 and B3GNT3 expression in PCSCs regulate stemness through modulating CSC markers.
Highlights
Glycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC)
TM treatment resulted in altered N-linked glycosylation of stem cell markers (ESA and CDDv6) and BAG treatment resulted in global variation of Tn antigen, O-linked glycosylation, as detected by VVA staining in PC cells (Additional file 2: Figure S1e and S1f )
Because cancer stem cells (CSCs) have been implicated in tumor initiation and metastasis, we examined if expression of tumor-associated carbohydrate antigens (TACAs), such as Tn, T, sTn, sialyl Lewis x (sLex), and sialyl Lewis a (sLea), is altered in CSCs/Side Population (SP) cells of SW1990 and Capan1 PC cells
Summary
Glycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC). Emerging evidences indicate significant involvement of cancer stem cells (CSCs) in PC aggressiveness. The importance of glycosylation in pancreatic cancer stem cells (PCSCs) is yet to be addressed. We evaluated the potential role of glycosylation in maintenance of stemness of PCSCs. Pancreatic cancer is a highly lethal malignancy, estimated to become the second-leading cause of cancer related deaths by 2030 [1]. CSCs are a rare, small subset of cells with the capacity to give rise to full tumor mass. They have a self-renewal capacity and undergo asymmetric or symmetric cell division producing a heterogeneous cell population [5,6,7]. CD133+CXCR4+ was identified as a subpopulation of PCSCs that mediates tumor metastasis in PC [8, 10, 11]
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