Quantitative site- and structure-specific N-glycoproteomics characterization of differential N-glycosylation in MCF-7/ADR cancer stem cells

Abstract

Background: Cancer stem cells (CSCs) are reported to be responsible for tumor initiation, progression, metastasis, and therapy resistance where P-glycoprotein (P-gp) as well as other glycoproteins are involved. Identification of these glycoprotein markers is critical for understanding the resistance mechanism and developing therapeutics. Methods: In this study, we report our comparative and quantitative site-and structure-specific N-glycoproteomics study of MCF-7/ADR cancer stem cells (CSCs) vs. MCF-7/ADR cells. With zic-HILIC enrichment, isotopic diethyl labeling, RPLC-MS/MS (HCD) analysis and GPSeeker DB search, differentially expressed N-glycosylation was quantitatively characterized at the intact N-glycopeptide level. Results: 4016 intact N-glycopeptides were identified with spectrum-level FDR ≤ 1%. With the criteria of ≥ 1.5 fold change and p value < 0.05, 247 intact N-glycopeptides were found differentially expressed in MCF-7/ADR CSCs as putative markers. Raw data are available via ProteomeXchange with identifier PXD013836. Conclusions: Quantitative site-and structure-specific N-glycoproteomics characterization may help illustrate the cell stemness property.

N-Glycoproteomics Study of Putative N-Glycoprotein Biomarkers of Drug Resistance in MCF-7/ADR Cells

“…and CSC N-glycosylation markers (centrosome-associated protein 350 CE350, zinc finger protein GLI1, CD63 antigen and CD13 etc.) were reported in our previous studies (Wang et al 2020b;Xu et al 2020).…”

“…In comparison of DEGPs in MCF-7 CSCs (relative to MCF-7 cells) (Wang et al 2019) with those in MCF-7/ADR CSCs (relative to MCF-7/ADR cells) (Xu et al 2020), intact N-glycopeptide SLSNSTAR_N2H5F0S0 (P50454, SERPH_ HUMAN, N120) showed the same up-regulation with ratios of 4.23 ± 0.71 and 3.80 ± 0.55, respectively. We found that the expression level of intact N-glycopeptides SLSN-STAR_N2Hx (x = 5-9) was decreased progressively from up-regulation to down-regulation in MCF-7/ADR CSCs (relative to MCF-7/ADR cells) (Xu et al 2020).…”

“…In comparison of DEGPs in MCF-7 CSCs (relative to MCF-7 cells) (Wang et al 2019) with those in MCF-7/ADR CSCs (relative to MCF-7/ADR cells) (Xu et al 2020), intact N-glycopeptide SLSNSTAR_N2H5F0S0 (P50454, SERPH_ HUMAN, N120) showed the same up-regulation with ratios of 4.23 ± 0.71 and 3.80 ± 0.55, respectively. We found that the expression level of intact N-glycopeptides SLSN-STAR_N2Hx (x = 5-9) was decreased progressively from up-regulation to down-regulation in MCF-7/ADR CSCs (relative to MCF-7/ADR cells) (Xu et al 2020). Interestingly, the decreased expression level of SLSNSTAR_N2Hx (x = 5-9) was gradually levelled off in our present system of MCF-7/ADR cells vs. MCF-7 cells; from N2H5 to N2H9, fold changes were 0.27 ± 0.03, 0.49 ± 0.08, 0.76 ± 0.08, 0.92 ± 0.08 and 1.09 ± 0.29, respectively (Fig.…”

“…Hence, it is still urgent to figure out the mechanisms of drug resistance. In this regard, intact N-glycoproteins about the development of drug resistance, either identified or quantified in current and previous studies (Wang et al 2019(Wang et al , 2020bXu et al 2020;Xue et al 2020), were discussed.…”

Abnormal Glycosylation of Cancer Stem Cells and Targeting Strategies

“…Moreover, hypersialylation in cancer cells promotes cell migration (18) and apoptosis resistance (19), which lead to tumor growth and poor prognosis (16). CSCs may be further distinguished from differentiated cancer cells by the expression of carbohydrate antigens (20,21), and the Nglycosylation of glycoproteins of CSCs has been related to drug resistance mechanisms (22). Besides, abnormal glycosylation has been shown to regulate signaling pathways and markers involved in conservation of CSCs, such as EpCAM glycosylation on cell adhesion (23) and EMT in breast cancer, or N-glycosylation on MAPK and PI3/Akt pathway (24).…”