N-cadherin upregulation mediates adaptive radioresistance in glioblastoma
Satoru Osuka, … , Christopher D. Willey, Erwin G. Van Meir
Satoru Osuka, … , Christopher D. Willey, Erwin G. Van Meir
Published March 15, 2021
Citation Information: J Clin Invest. 2021;131(6):e136098. https://doi.org/10.1172/JCI136098.
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Research Article Oncology

N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

Abstract

Glioblastoma (GBM) is composed of heterogeneous tumor cell populations, including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance in GBM, we generated radioresistant human and mouse GSCs by exposing them to repeat cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by a reduction in cell proliferation and an increase in cell-cell adhesion and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of β-catenin at the cell surface, which suppressed Wnt/β-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin, a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation.

Authors

Satoru Osuka, Dan Zhu, Zhaobin Zhang, Chaoxi Li, Christian T. Stackhouse, Oltea Sampetrean, Jeffrey J. Olson, G. Yancey Gillespie, Hideyuki Saya, Christopher D. Willey, Erwin G. Van Meir

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Figure 6

Enhanced N-cad elevates Clusterin expression and protects against radiation-induced apoptosis.

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Enhanced N-cad elevates Clusterin expression and protects against radiat...
(A) Heatmap comparing 9 gene mRNA expression measured by RNA-seq analysis in untreated mGS cells (Ctrl), in mGS cells after 6–12 cycles of fractionated irradiation with 5 Gy (Ct, 30 Gy, 45 Gy, and 60 Gy total dose), in mGS or mGSRR N-cad–KO cells (mGS N-cad–KO #1, #2, and mGSRR N-cad–KO), with or without N-cad reconstitution (OE). Two independent replicates per cell line (n = 2). (B and C) Western blot showing expression of N-cad and Clu are gradually increased following mouse GS (B) and human MGG4 (C) adaptation to fractionated irradiation. (D) Western blot showing expression of Clu is suppressed by N-cad–KO and restored by stable N-cad transfection in mGS cells. (E) Western blot showing expression of Clu is suppressed by shRNA-mediated knockdown of N-cad in JX39-RT radioresistant PDX cells. (F) CDH2 and CLU mRNA expression correlate in GBM (TCGA database). (G) Clonogenic survival assay for mGS cells transfected with control or Clu expression vectors and mGSRR cells transfected with shCtrl or shClu expression vectors with or without a single dose (3 Gy) of IR. Left: representative images of colonies formed by surviving cells 13 days after irradiation. Scale bar: 10 mm. Right: quantification of the fraction of surviving colony-forming cells. ***P < 0.001, Tukey’s HSD test. (H) ELISA assay showing that Clu secretion is remarkably increased by mGSRR compared with mGS cells and this is strongly suppressed by N-cad knockout. **P < 0.01, ***P < 0.001, Tukey’s HSD test. (I) Western blot showing that suppression of Clu expression by shRNA or as a result of N-cad knockout increases PARP cleavage in mGSRR cells. (J) Kaplan-Meier curve shows that increased CLU mRNA expression is correlated with poor outcome in the TCGA-GBM data set. High and low are defined as top and bottom 15%. All blots show representative images (n = 3).