title: Glut-1 as a therapeutic target: increased chemoresistance and HIF-1-independent link with cell turnover is revealed through COMPARE analysis and metabolomic studies.
abstract: The facilitative glucose transporter Glut-1 is overexpressed and confers poor prognosis in a wide range of solid tumours. The peri-necrotic pattern of expression often seen in human tumour samples is linked with its transcriptional control in hypoxic conditions by hypoxia-inducible factor HIF-1 or through a reduced rate of oxidative phosphorylation. Hypoxia-regulated genes offer promise as novel therapeutic targets as a means of preventing the proliferation and eventual metastatic spread of tissue originating from residual chemically and radio resistant hypoxic cells that have survived treatment. Inhibiting the expression or functionality of Glut-1 may be a way of specifically targeting hypoxic cells within the tumour that depend upon a high rate of glucose uptake for anaerobic glycolysis. We used an array of formalin-fixed, paraffin-embedded samples of the NCI-60 panel of cell lines to carry out immunohistochemical detection of Glut-1 and to select possible candidate lead compounds by COMPARE analysis with agents from the NCI diversity screen, which may work via inhibition of Glut-1 or Glut-1-dependent processes. "Positive" COMPARE hits were mostly conjugated Pseudomonas toxins binding the epidermal growth factor receptor (EGFR). However, correlations with standard anticancer agents were virtually all negative, indicating a link between Glut-1 and chemoresistance. MTT proliferation assays carried out using stable, Glut-1 overexpressing cell lines generated from the bladder EJ138, human fibrosarcoma HT 1080 and the hepatoma wild type Hepa and HIF-1B-deficient c4 tumour cell lines revealed a cell line-dependent increase in chemoresistance to dacarbazine, vincristine and the bioreductive agent EO9 in Glut-1 overexpressing EJ138 relative to WT and empty vector controls. Metabolomic analysis ((31)P-MRS and (1)H MRS) carried out using cell lysates and xenografts generated from Glut-1 overexpressing Hepa and c4 cell lines showed higher glucose levels in Glut-1 overxpressing c4 relative to parental tumour extracts occurred in the absence of an increase in lactate levels, which were in turn significantly higher in the Glut-1 overexpressing Hepa xenografts. This implies that Glut-1 over-expression without a co-ordinate increase in HIF-1-regulated glycolytic enzymes increases glucose uptake but not the rate of glycolysis. Glut-1 overexpressing xenografts also showed higher levels of phosphodiester (PDE), which relates to the metabolite turnover of phospholipids and is involved in membrane lipid degradation, indicating a mechanism by which Glut-1 may increase cell turnover.
authors.affiliation: Tumour Metabolism and Therapeutics Group, School of Pharmacy and Chemistry, Liverpool John Moores University, Liverpool L3 3AF, UK. a.r.evans@ljmu.ac.uk
authors.forename: Andrew
authors.initials: A
authors.lastname: Evans
authors.forename: Victoria
authors.initials: V
authors.lastname: Bates
authors.forename: Helen
authors.initials: H
authors.lastname: Troy
authors.forename: Stephen
authors.initials: S
authors.lastname: Hewitt
authors.forename: Susan
authors.initials: S
authors.lastname: Holbeck
authors.forename: Yuen-Li
authors.initials: YL
authors.lastname: Chung
authors.forename: Roger
authors.initials: R
authors.lastname: Phillips
authors.forename: Marion
authors.initials: M
authors.lastname: Stubbs
authors.forename: John
authors.initials: J
authors.lastname: Griffiths
authors.forename: Rachel
authors.initials: R
authors.lastname: Airley
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D030541:Databases, Genetic; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004354:Drug Screening Assays, Antitumor; D005455:Fluorescent Antibody Technique; D051272:Glucose Transporter Type 1 / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000502:physiology*; D007150:Immunohistochemistry; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy / Q000473:pathology; D009682:Magnetic Resonance Spectroscopy; D008660:Metabolism; D016612:Paraffin Embedding; D013778:Tetrazolium Salts; D013844:Thiazoles; D016707:Tissue Fixation; D023041:Xenograft Model Antitumor Assays
chemical_list: D051272:Glucose Transporter Type 1; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D013778:Tetrazolium Salts; D013844:Thiazoles; D049452:Green Fluorescent Proteins; C022616:thiazolyl blue