Glycosylation of Cancer Stem Cells: Function in Stemness, Tumorigenesis, and Metastasis

Aberrant glycosylation plays a critical role in tumor aggressiveness, progression, and metastasis. Emerging evidence associates cancer initiation and metastasis to the enrichment of cancer stem cells (CSCs). Several universal markers have been identified for CSCs characterization; however, a specific marker has not yet been identified for different cancer types. Specific glycosylation variation plays a major role in the progression and metastasis of different cancers. Interestingly, many of the CSC markers are glycoproteins and undergo differential glycosylation. Given the importance of CSCs and altered glycosylation in tumorigenesis, the present review will discuss current knowledge of altered glycosylation of CSCs and its application in cancer research

Novel role of O-glycosyltransferases GALNT3 and B3GNT3 in the self-renewal of pancreatic cancer stem cells

Abstract Background 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. 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. Methods Effect 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 Kras G12D ; Pdx-1-Cre (KC) and Kras G12D ; p53 R172H ; 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. Results Inhibition 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. Conclusions Taken 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

Rhizoctonia bataticola lectin (RBL) induces caspase-8-mediated apoptosis in human T-cell leukemia cell lines but not in normal CD3 and CD34 positive cells.

We have previously demonstrated immunostimulatory activity of a fungal lectin, Rhizoctonia bataticola lectin (RBL), towards normal human peripheral blood mononuclear cells. The present study aimed to explore the anticancer activities of RBL using human leukemic T-cell lines, Molt-4, Jurkat and HuT-78. RBL exhibited significant binding (>90%) to the cell membrane that was effectively inhibited by complex glycoproteins such as mucin (97% inhibition) and asialofetuin (94% inhibition) but not simple sugars such as N-acetyl-D-galactosamine, glucose and sucrose. RBL induced a dose and time dependent inhibition of proliferation and induced cytotoxicity in the cell lines. The percentage of apoptotic cells, as determined by hypodiploidy, was 33% and 42% in Molt-4 and Jurkat cells, respectively, compared to 3.11% and 2.92% in controls. This effect was associated with a concomitant decrease in the G0/G1 population. Though initiator caspase-8 and -9 were activated upon exposure to RBL, inhibition of caspase-8 but not caspase-9 rescued cells from RBL-induced apoptosis. Mechanistic studies revealed that RBL induced cleavage of Bid, loss of mitochondrial membrane potential and activation of caspase-3. The expression of the anti-apoptotic proteins Bcl-2 and Bcl-X was down regulated without altering the expression of pro-apoptotic proteins--Bad and Bax. In contrast to leukemic cells, RBL did not induce apoptosis in normal PBMC, isolated CD3+ve cells and undifferentiated CD34+ve hematopoietic stem and progenitor cells (HSPCs). The findings highlight the differential effects of RBL on transformed and normal hematopoietic cells and suggest that RBL may be explored for therapeutic applications in leukemia

Effect of RBL on different phases of cell cycle.

<p>(A) Molt -4 and Jurkat cells, treated with RBL, were stained with PI and acquired on FL2-A channel of flow cytometer equipped with 488nm laser. The X-axis represents the DNA content of the cells and the Y-axis represents the cell number. The graph depicts the percentage of Molt-4 (B) and Jurkat (C) cells in different phases of cell cycle. The data is mean ±SE of three independent experiments. *p<0.05 significant difference between untreated and RBL treated cells.</p

Effect of RBL on activation of caspases.

<p>Molt-4 cells were treated with 5 µg/ml RBL for different time periods and activities of caspase-8 (A) and caspase-9 (B) were assessed by colorimetric assay using specific substrates Ac-IETD-pNa and Ac-LEHD-pNa respectively. The graph depicts fold increase in caspase activity with respect to 0 h controls (activity at 0 h was considered as 1). The data is mean±SE values from three independent experiments. *p value <0.05 in comparison with 0 h controls. (C) Molt-4 cells were treated with RBL (5 µg/ml) for 12 h and activity of caspase-3 was determined by flow cytometry analysis using FITC tagged caspase–3 inhibitor (blue line). Cells pretreated with caspase-3 inhibitor ZVAD-FMK (red line) and untreated cells (green line) were used as controls. The histoplot is representative of three similar experiments.</p

Effect of caspase-8 inhibition on RBL-induced apoptosis.

<p>Molt-4 (A) and Jurkat cells (B) were treated with RBL(5 µg/ml) for 12 h in the presence or absence of caspase inhibitors(40 µM) zVAD-FMK (pan caspase inhibitor), zIETD-FMK (caspase-8 inhibitor), or zLEHD-FMK (caspase-9 inhibitor) and viability was assessed by MTT assay. The graphs depict mean±SE values from three independent experiments. *p value <0.05 in comparison with untreated controls. Molt-4 (C) and Jurkat (D) cells were treated with RBL (5 µg/ml) for 12 h in the presence or absence of caspase-8 inhibitor (zIETD-FMK) and caspase-3 activation was assessed using flow cytometry. The overlay depicts profile of untreated cells (black line), cells pretreated with caspase-3 inhibitor followed by RBL exposure (red line) and cells treated with RBL alone (blue line).</p

Effect of RBL on proliferation and viability of leukemic cell lines-.

<p>Molt-4 (A) and Jurkat (B) cells were exposed to serial concentrations of RBL for 72 h and proliferation was determined by tritiated thymidine incorporation assay. The percentage cell proliferation was calculated by considering the counts per minute of untreated control cells as 100. Molt-4(C) and Jurkat (D) cell lines were exposed to serial concentrations of RBL for different time periods and cell viability was assessed by MTT assay. The absorbance value of untreated cells was considered as 100 to calculate percent viable cell number. The values presented in the graph are mean±SE of three independent experiments done in triplicates. *p<0.05 difference compared to untreated cells.</p

Effect of RBL on HUT-78 cells.

<p>(A) HUT-78 cells were exposed to RBL (5 µg/ml) and cell viability was assessed by MTT assay. The absorbance of untreated cells was considered as 100 to calculate percent viable cell number. The values presented in the graph are mean±SE of three independent experiments done in triplicates. *p<0.05 difference compared to untreated cells. (B) Cells treated with RBL were stained with PI and cell cycle analysis was done with data acquired on FL2-A channel. The X-axis represents the DNA content of the cells and the Y-axis represents the cell number. (C) The graph depicts the percentage of cells in different phases of cell cycle. The graph represents mean ±SE of three independent experiments. *p<0.05 significant difference between untreated and RBL-treated cells.</p

RBL induces apoptosis of Molt-4 and Jurkat cells.

<p>(A) Molt-4 cells were treated with RBL (5 µg/ml) for 6, 12 and 24 h followed by Annexin- V-FITC and PI staining. The X-axis depicts Annexin-V positive cells and Y- axis depicts PI positive cells. The numbers in each quadrant represent percent positive cells. Dot plots are representative of three similar experiments. (B) The graph represents % apoptosis (Annexin-V-positive cells) mean±SE of three independent experiments. *p<0.05 indicates significant difference between treated and untreated cells. (C) Whole cell lysates of Molt-4 and Jurkat cells were exposed to RBL (1.25 to 5 µg/ml) for 24 h and probed with anti-PARP antibody. The blot shows total and cleaved PARP. The data is representative of two similar experiments.</p