α2,6 Sialylation mediated by ST6GAL1 promotes glioblastoma growth
Sajina GC, Kaysaw Tuy, Lucas Rickenbacker, Robert Jones, Asmi Chakraborty, C. Ryan Miller, Elizabeth A. Beierle, Vidya Sagar Hanumanthu, Anh N. Tran, James A. Mobley, Susan L. Bellis, Anita B. Hjelmeland
Sajina GC, Kaysaw Tuy, Lucas Rickenbacker, Robert Jones, Asmi Chakraborty, C. Ryan Miller, Elizabeth A. Beierle, Vidya Sagar Hanumanthu, Anh N. Tran, James A. Mobley, Susan L. Bellis, Anita B. Hjelmeland
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Research Article Cell biology Oncology

α2,6 Sialylation mediated by ST6GAL1 promotes glioblastoma growth

Abstract

One of the least-investigated areas of brain pathology research is glycosylation, which is a critical regulator of cell surface protein structure and function. β-Galactoside α2,6-sialyltransferase (ST6GAL1) is the primary enzyme that α2,6 sialylates N-glycosylated proteins destined for the plasma membrane or secretion, thereby modulating cell signaling and behavior. We demonstrate a potentially novel, protumorigenic role for α2,6 sialylation and ST6GAL1 in the deadly brain tumor glioblastoma (GBM). GBM cells with high α2,6 sialylation exhibited increased in vitro growth and self-renewal capacity and decreased mouse survival when orthotopically injected. α2,6 Sialylation was regulated by ST6GAL1 in GBM, and ST6GAL1 was elevated in brain tumor-initiating cells (BTICs). Knockdown of ST6GAL1 in BTICs decreased in vitro growth, self-renewal capacity, and tumorigenic potential. ST6GAL1 regulates levels of the known BTIC regulators PDGF Receptor β (PDGFRB), Activated Leukocyte Cell Adhesion Molecule, and Neuropilin, which were confirmed to bind to a lectin-recognizing α2,6 sialic acid. Loss of ST6GAL1 was confirmed to decrease PDGFRB α2,6 sialylation, total protein levels, and the induction of phosphorylation by PDGF-BB. Thus, ST6GAL1-mediated α2,6 sialylation of a select subset of cell surface receptors, including PDGFRB, increases GBM growth.

Authors

Sajina GC, Kaysaw Tuy, Lucas Rickenbacker, Robert Jones, Asmi Chakraborty, C. Ryan Miller, Elizabeth A. Beierle, Vidya Sagar Hanumanthu, Anh N. Tran, James A. Mobley, Susan L. Bellis, Anita B. Hjelmeland

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Figure 1

α2,6 Sialylation increases GBM growth and self-renewal.

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α2,6 Sialylation increases GBM growth and self-renewal. (A) Schematic of...
(A) Schematic of SNA, lectin with high affinity for α2,6 sialic acid, tagged with FITC as used for flow cytometry. (B) Representative histogram using SNA-FITC for FACS to sort SNAhi or α2,6 sialylationhi (highest 10% intensity) and SNAlo or α2,6 sialylationlo (lowest 10% intensity) cells. A total of 1,000 α2,6 sialylationhi versus α2,6 sialylationlo cells isolated from (C) D456 and (D) JX39 GBM PDXs were directly plated during FACS, and growth was measured over time using CellTiter-Glo 2.0 (luminescence, RLU). Individual data points are shown with the error bars as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, 2-way ANOVA with Tukey’s multiple comparisons test. The experiments were repeated in 3 independent biological replicates. Data from 1 representative experiment are shown. Differences in self-renewal and BTIC frequencies were determined using in vitro limiting dilution assays with α2,6 sialylationhi versus α2,6 sialylationlo cells isolated from (E) D456 and (F) JX39 GBM PDXs. Each group was plated in decreasing number of cells (100, 50, 10, 5, and 1 cell per well). Extreme limiting dilution analysis (ELDA) was done using the software (http://bioinf.wehi.edu.au/software/elda/). P values were calculated from χ2 analysis of group comparisons. The experiments were repeated in 3 independent biological replicates. Data from 1 representative experiment are shown. (G) Kaplan-Meier survival curves for BALB/c nu/nu mice injected orthotopically with 2,500 α2,6 sialylationhi or α2,6 sialylationlo cells isolated from D456 PDX cells and euthanized upon development of neurological signs. P value was calculated using log-rank (Mantel-Cox) test. (H) Representative histological images of tumors stained with H&E support the presence of brain tumors in mice with neurological signs. Top panels: Image objective = 1.25×; scale bar: 1.0 mm. Bottom panels: Image objective = 20×; scale bar: 0.1 mm.