title: Silencing gastrin-releasing peptide receptor suppresses key regulators of aerobic glycolysis in neuroblastoma cells.
abstract: BACKGROUND
  Under normoxic conditions, cancer cells use aerobic glycolysis as opposed to glucose oxidation for energy production; this altered metabolism correlates with poor outcomes in neuroblastoma. Hypoxia-inducible factor-1 alpha (HIF-1α) and pyruvate dehydrogenase kinase 4 (PDK4) regulate aerobic glycolysis, while pyruvate dehydrogenase phosphatase 2 (PDP2) promotes glucose oxidation. Here, we sought to determine whether gastrin-releasing peptide receptor (GRP-R) signaling regulates glucose metabolism.
  
  
  METHODS
  Neuroblastoma cell lines, BE(2)-C and SK-N-AS, were used. PCR microararay for glucose metabolism was performed on GRP-R silenced cells. Target protein expression was validated using Western blotting and VEGF ELISA. Cobalt chloride (CoCl2 ) was used to induce chemical hypoxia. Efficacy of targeting PDK regulation in neuroblastoma was assessed using dichloroacetate (DCA) by conducting cell viability assays and Western blotting for apoptotic markers.
  
  
  RESULTS
  Silencing GRP-R decreased HIF-1α expression and blocked VEGF expression and secretion in both normoxic and CoCl2 induced hypoxia. PCR array analysis identified that GRP-R silencing reduced PDK4 and increased PDP2 mRNA expression. These findings were validated by Western blotting. CoCl2 induced hypoxia increased VEGF secretion, HIF-1α, and PDK4 expression. PDK4 silencing decreased HIF-1α expression and VEGF expression and secretion. DCA treatment decreased BE(2)-C and SK-N-AS proliferation while promoting cell death. GRP-R silencing and DCA treatment synergistically halted BE(2)-C proliferation.
  
  
  CONCLUSIONS
  We report that GRP-R regulates glucose metabolism in neuroblastoma by modulating HIF-1α, PDK4 and PDP2. PDK4 regulates glucose metabolism, in part, via regulation of HIF-1α. Synergistic consequences of GRP-R inhibition and DCA treatment may suggest a novel therapeutic strategy for the treatment of aggressive neuroblastoma.
authors: Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, Tennessee.
authors: Eric J
authors: EJ
authors: Rellinger
authors: Carmelle
authors: C
authors: Romain
authors: SunPhil
authors: S
authors: Choi
authors: Jingbo
authors: J
authors: Qiao
authors: Dai H
authors: DH
authors: Chung
mesh_terms: D016587:Antimutagenic Agents / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D003035:Cobalt / Q000494:pharmacology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006019:Glycolysis; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D018004:Receptors, Bombesin / Q000096:biosynthesis* / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
chemical_list: D016587:Antimutagenic Agents; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009363:Neoplasm Proteins; C000631432:PDK4 protein, human; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D018004:Receptors, Bombesin; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D003035:Cobalt; D017346:Protein Serine-Threonine Kinases; C018021:cobaltous chloride
keywords: GRP-R; HIF-1α; PDK4; VEGF; glycolysis; neuroblastoma
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