Cancer stem cells (CSCs) play an important role in disease recurrence after radiation treatment as a result of intrinsic properties such as high DNA repair capability and antioxidative capacity. It is unclear, however, how CSCs further adapt to escape the toxicity of the repeated irradiation regimens used in clinical practice. Here, we have exposed a population of murine glioma stem cells (GSCs) to fractionated radiation in order to investigate the associated adaptive changes, with the ultimate goal of identifying a targetable factor that regulates acquired radioresistance. We have shown that fractionated radiation induces an increase in IGF1 secretion and a gradual upregulation of the IGF type 1 receptor (IGF1R) in GSCs. Interestingly, IGF1R upregulation exerts a dual radioprotective effect. In the resting state, continuous IGF1 stimulation ultimately induces downregulation of Akt/extracellular-signal-regulated kinases (ERK) and FoxO3a activation, which results in slower proliferation and enhanced self-renewal. In contrast, after acute radiation, the abundance of IGF1R and increased secretion of IGF1 promote a rapid shift from a latent state toward activation of Akt survival signaling, protecting GSCs from radiation toxicity. Treatment of tumors formed by the radioresistant GSCs with an IGF1R inhibitor resulted in a marked increase in radiosensitivity, suggesting that blockade of IGF1R signaling is an effective strategy to reverse radioresistance. Together, our results show that GSCs evade the damage of repeated radiation not only through innate properties but also through gradual inducement of resistance pathways and identify the dynamic regulation of GSCs by IGF1R signaling as a novel mechanism of adaptive radioprotection.

Extracellular free amino acids contribute to the interaction between a tumor and its microenvironment through effects on cellular metabolism and malignant behavior. System xc(–) is composed of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. Here, we show that the EGFR interacts with xCT and thereby promotes its cell surface expression and function in human glioma cells. EGFR-expressing glioma cells manifested both enhanced antioxidant capacity as a result of increased cystine uptake, as well as increased glutamate, which promotes matrix invasion. Imaging mass spectrometry also revealed that brain tumors formed in mice by human glioma cells stably overexpressing EGFR contained higher levels of reduced glutathione compared with those formed by parental cells. Targeted inhibition of xCT suppressed the EGFR-dependent enhancement of antioxidant capacity in glioma cells, as well as tumor growth and invasiveness. Our findings establish a new functional role for EGFR in promoting the malignant potential of glioma cells through interaction with xCT at the cell surface. Cancer Res; 76(10); 2954–63. ©2016 AACR.

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.

Gold deposition with diagonal angle towards boehmite-based nanostructure creates random arrays of horse-bean-shaped nanostructures named gold-nanofève (GNF). GNF generates many electromagnetic hotspots as surface-enhanced Raman spectroscopy (SERS) excitation sources, and enables large-area visualization of molecular vibration fingerprints of metabolites in human cancer xenografts in livers of immunodeficient mice with sufficient sensitivity and uniformity. Differential screening of GNF-SERS signals in tumours and those in parenchyma demarcated tumour boundaries in liver tissues. Furthermore, GNF-SERS combined with quantum chemical calculation identified cysteine-derived glutathione and hypotaurine (HT) as tumour-dominant and parenchyma-dominant metabolites, respectively. CD44 knockdown in cancer diminished glutathione, but not HT in tumours. Mechanisms whereby tumours sustained HT under CD44-knockdown conditions include upregulation of PHGDH, PSAT1 and PSPH that drove glycolysis-dependent activation of serine/glycine-cleavage systems to provide one-methyl group for HT synthesis. HT was rapidly converted into taurine in cancer cells, suggesting that HT is a robust anti-oxidant for their survival under glutathione-suppressed conditions.

Glioblastomas have been shown to rely on glycolysis as an energy source. However, recent evidence suggests that at least a subset of glioma cells with stem cell–like properties can thrive on oxidative phosphorylation. It remains unclear whether both metabolic phenotypes support tumor propagation, if they are independent, and how stable they are. The present study investigated these questions with the use of isogenic murine glioma stem cells (GSCs). GSCs were established from tumors formed by Ink4a/Arf–null, H-Ras^V12-expressing glioma-initiating cells that differed in extracellular acidification potential. Metabolic characteristics of GSCs were determined by measurement of glucose, oxygen, and glutamine uptake, ATP content, and lactate production. Effects of metabolic inhibitors and changes in oxygen or nutrient availability on lactate production and tumorsphere growth were also determined. GSCs were found either to consume more glucose and produce more lactate or to consume more oxygen and maintain a higher ATP content depending on the metabolic characteristics of the tumor cells of origin. The latter, mitochondrial-type GSCs increased lactate production after treatment with the oxidative phosphorylation inhibitor oligomycin or phenformin. Exposure to hypoxia also increased lactate production and expression of glycolysis-related enzymes and metabolites in mitochondrial-type GSCs in a reversible manner. Both glycolytic and mitochondrial-type energy production can sustain tumor propagation by isogenic GSCs. Whereas both phenotypes can be independent and stable, cells that rely on oxidative phosphorylation can also switch to a more glycolytic phenotype in response to metabolic stress, suggesting that plasticity is a further characteristic of GSC metabolism.