title: Mitochondrial mutations contribute to HIF1alpha accumulation via increased reactive oxygen species and up-regulated pyruvate dehydrogenease kinase 2 in head and neck squamous cell carcinoma.
abstract: OBJECTIVE
  Mitochondrial mutations have been identified in head and neck squamous cell carcinoma (HNSCC), but the pathways by which phenotypic effects of these mutations are exerted remain unclear. Previously, we found that mitochondrial ND2 mutations in primary HNSCC increased reactive oxygen species (ROS) and conferred an aerobic, glycolytic phenotype with HIF1alpha accumulation and increased cell growth. The purpose of the present study was to examine the pathways relating these alterations.
  
  
  METHODS
  Mitochondrial mutant and wild-type ND2 constructs were transfected into oral keratinocyte immortal cell line OKF6 and head and neck cancer cell line JHU-O19 and established transfectants. The protein levels of HIF1alpha, pyruvate dehydrogenease (PDH), phosphorylated PDH, and pyruvate dehydrogenease kinase 2 (PDK2), together with ROS generation, were compared between the mutant and the wild type. Meanwhile, the effects of small molecule inhibitors targeting PDK2 and mitochondria-targeted catalase were evaluated on the ND2 mutant transfectants.
  
  
  RESULTS
  We determined that ND2 mutant down-regulated PDH expression via up-regulated PDK2, with an increase in phosphorylated PDH. Inhibition of PDK2 with dichloroacetate decreased HIF1alpha accumulation and reduced cell growth. Extracellular treatment with hydrogen peroxide, a ROS mimic, increased PDK2 expression and HIF1alpha expression, and introduction of mitochondria-targeted catalase decreased mitochondrial mutation-mediated PDK2 and HIF1alpha expression and suppressed cell growth.
  
  
  CONCLUSIONS
  Our findings suggest that mitochondrial ND2 mutation contributes to HIF1alpha accumulation via increased ROS production, up-regulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in HIF1alpha stabilization. This may provide insight into a potential mechanism, by which mitochondrial mutations contribute to HNSCC development.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA.
authors.forename: Wenyue
authors.initials: W
authors.lastname: Sun
authors.forename: Shaoyu
authors.initials: S
authors.lastname: Zhou
authors.forename: Steven S
authors.initials: SS
authors.lastname: Chang
authors.forename: Thomas
authors.initials: T
authors.lastname: McFate
authors.forename: Ajay
authors.initials: A
authors.lastname: Verma
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Califano
mesh_terms: D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008819:Mice, Nude; D008928:Mitochondria / Q000235:genetics*; D009154:Mutation; D009245:NADH Dehydrogenase / Q000235:genetics* / Q000502:physiology; D009368:Neoplasm Transplantation; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017382:Reactive Oxygen Species
chemical_list: D004272:DNA, Mitochondrial; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C000631430:PDK2 protein, human; C000631435:Pdk2 protein, mouse; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017382:Reactive Oxygen Species; D009245:NADH Dehydrogenase; C521326:NADH dehydrogenase subunit 2, human; D017346:Protein Serine-Threonine Kinases