Published OnlineFirst July 7, 2014; DOI: 10.1158/1541-7786.MCR-14-0106-T Molecular Cancer Cell Death and Survival Research Glucose-6–phosphatase Is a Key Metabolic Regulator of Glioblastoma Invasion Sara Abbadi1, Julio J. Rodarte1, Ameer Abutaleb2, Emily Lavell1, Chris L. Smith1,3, William Ruff1, Jennifer Schiller4, Alessandro Olivi1, Andre Levchenko3, Hugo Guerrero-Cazares1, and Alfredo Quinones-Hinojosa1 Abstract Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear especially in a hypoxic tumor microenvironment. In this study, it was determined that glucose-6–phosphatase (G6PC/G6Pase) expression is elevated in GBM when compared with normal brain. Human-derived brain tumor–initiating cells (BTIC) use this enzyme to counteract glycolytic inhibition induced by 2-deoxy-D-glucose (2DG) and sustain malignant progression. Downregulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation). Collectively, these findings establish G6PC as a key enzyme with promalignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target. Implications: This study is the first to demonstrate a functional relationship between the critical gluconeogenic and glycogenolytic enzyme G6PC with the metabolic adaptations during GBM invasion. Visual Overview: http://mcr.aacrjournals.org/content/12/11/1547/F1.large.jpg. Mol Cancer Res; 12(11); 1547–59. Ó2014 AACR. Introduction brain, these cells are called brain tumor–initiating cells Glioblastoma (GBM) is the most aggressive primary brain (BTIC), cancer stem cells, or tumor-propagating cells (5, 6). cancer in adults (1, 2). The diffuse infiltrative nature of The rapid expansion of GBMs and other solid tumors causes them to frequently outgrow their blood supply leading GBMs makes their total surgical resection impossible, leading fi to poor prognosis and short survival (3). A growing body of to oxygen de ciency and nutrient deprivation (7, 8). As a evidence has demonstrated that cancer cells display cellular response, tumors undergo angiogenesis to increase their oxy- gen and nutrient supply (9). Antiangiogenic therapy in solid hierarchies with a subset of cells believed to be responsible fi for resistance to conventional cancer therapies and for pro- tumors shows bene cial effects associated with a reduction of motion of tumor growth (4). In cancers originating in the the vasculature and delayed tumor progression, but ultimately increases tumor hypoxia, and induces a treatment-resistant phenotype (10). These findings, which are unique to cancer 1Department of Neurosurgery and Oncology, Johns Hopkins University cells, are driven by a constant metabolic reprogramming that School of Medicine, Baltimore, Maryland. 2University of Maryland, School of Medicine, Baltimore, Maryland. 3Department of Biomedical Engineering, enhances their survival adaptation in response to hypoxia (11). Johns Hopkins University School of Medicine, Baltimore, Maryland. 4Bran- One well-established example of this reprogramming is the deis University, Waltham, Massachusetts. Warburg effect (preference for aerobic glycolysis), which has Note: Supplementary data for this article are available at Molecular Cancer been of particular interest in cancer cell metabolism (12). Research Online (http://mcr.aacrjournals.org/). However, the enhanced glycolysis seen with the Warburg Current address for A. Levchenko: Yale Systems Biology Institute, Yale effect cannot be completely functional under hypoxic condi- University, New Haven, Connecticut. tions wherein nutrient supply is insufficient. Thus, cancer cells Corresponding Authors: Alfredo Quinones-Hinojosa, Department of Neu- may additionally activate other metabolic processes, such as rosurgery and Oncology, Johns Hopkins University, 1550 Orleans Street, CRB-II, Room 247A, Baltimore, MD 21201. Phone: 410-502-2869; Fax: glycogen mobilization, to provide intermediates for their 410-502-7559; E-mail: [email protected]; and Hugo Guerrero-Cazares, enhanced, reprogramed glycolytic pathway (13). [email protected] Metabolic reprogramming allows BTICs to survive and doi: 10.1158/1541-7786.MCR-14-0106-T adapt to restricted nutrition conditions (14). Targeting such Ó2014 American Association for Cancer Research. adaptation mechanism, which is common to most cancer www.aacrjournals.org 1547 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst July 7, 2014; DOI: 10.1158/1541-7786.MCR-14-0106-T Abbadi et al. cells, could be a crucial therapeutic tool (15). Previously, we 24 hours and were given additional 24 hour to recover before demonstrated that glycolytic inhibition of BTICs with the selection in 0.5 mg/mL puromycin (Sigma) for a minimum glucose analog 2-deoxy-D-glucose (2DG) promoted neu- of 6 days. ronal commitment and decreased cell proliferation rate, inducing a less malignant phenotype (16). However, some Invasion assay BTICs are able to survive glycolytic inhibition and recover Boyden Transwell assays were used to compare invasive  4 their aggressive phenotype. The precise mechanism by capacity of BTICs among treatment groups. A total of 5 10 which BTICs counteract glycolytic inhibition remains cells in each treatment group were resuspended in correspond- m unknown. ing media. Of note, 500 L of cell suspension was seeded into m A key enzyme in the regulation of glucose homeostasis and the top well of a Boyden chamber (BD Biosciences; 8- m m the glycogenolytic pathway is the glucose-6–phosphatase pores); 700 L of media were added to the lower chamber. fi complex, which is located at the membrane of the endo- After 24 hours incubation at 37 C, cells were xed, stained, fi plasmic reticulum (17). Because it serves a physiologically and counted under light microscopy (10 elds/insert). All important role in the glycogenolytic pathway, we hypoth- treatment groups were done in triplicate; all experiments were esized that glucose-6–phosphatase is required for BTIC repeated three times. survival, and that targeting it will commit BTICs to cell death. Neurosphere assay In this work, we report a regain of promalignant char- Neurosphere culture was performed as previously fl  3 acteristics of BTICs following their recovery from glycolytic described by our group (20). Brie y, 2.5 10 BTICs fl inhibition. We then identify that this recovery capacity is were plated in uncoated T25 asks in a total volume of 5 mL abolished when the hepatic isoform of glucose-6-phospha- of control media. Size and number of spheres were measured tase (G6PC) is inhibited. Furthermore, knocking down after 14 days of culture and analyzed. G6PC is sufficient to decrease the migratory and proliferative capacity of BTICs. These findings seem to be related to the Migration assay alterations in the glycogen metabolism of brain cancer cells. Migration of BTICs was assessed using topographic nanopatterned substratum, which consists of parallel ridges Materials and Methods 350-nm wide, 500-nm high, spaced 1.5-mm apart, fabricat- Cell culture ed onto glass coverslips as previously described by our group – All protocols in this study have been approved by the (21 23). Johns Hopkins Hospital Institutional Review Board. Pri- mary cultures of human fetal–derived astrocytes were Tumor xenografts in nude mice obtained as described previously (18, 19). Adult GBM tissue All animal protocols were approved by the Johns Hopkins  5 samples were collected after written informed consent. Animal Care and Use Committee. A total of 5 10 GBM1 m Patient characteristics are described in Supplementary Table BTICs were resuspended in 2 L of media and injected S1. Cell lines were tested and authenticated by the GRCF at intracranially into each of 25 male nude mice (Coordinates Johns Hopkins. Control media consisted of DMEM F12 AP 1.34; ML 1.5; and DV 3.5) as previously described by (þ) glutamine (Life Technologies), BIT9500 10% (STEM- our group (24). CELL Technologies) for a final glucose concentration of 25 mmol/L, and 20 ng/mL EGF and bFGF (basic fibroblast Results growth factor; Pepro Tech). 2DG-treated groups were BTICs escape glycolytic inhibition with an aggressive cultured for 18 hours in low-glucose media consisting of phenotype DMEM no-glucose (þ) glutamine (Life Technologies), with We performed all experiments using BTICs derived from BIT9500 (10%), 20 ng/mL EGF and bFGF (Pepro Tech) intraoperative GBM samples. The capacity of our BTICs to and 2DG 25 mmol/L (Sigma-Aldrich). Recovery groups form spheres in suspension, differentiate upon growth factor were obtained by replacing 2DG-supplemented media (after withdrawal, and form orthotopic tumors in animal models 18 hours) with control media for 72 hours. Of note, 0.1 was previously described (Supplementary Fig. S1 and Sup- mmol/L chlorogenic (CHL) acid (Sigma-Aldrich) was added plementary Table S1; refs. 20, 25, 26). To recapitulate the to recovery media in the recoveryþCHL groups. CP-91149 hypoxic tumor