title: Activation of AKT by hypoxia: a potential target for hypoxic tumors of the head and neck.
abstract: BACKGROUND
  Only a minority of cancer patients benefits from the combination of EGFR-inhibition and radiotherapy in head and neck squamous cell carcinoma (HNSCC). A potential resistance mechanism is activation of EGFR and/or downstream pathways by stimuli in the microenvironment. The aim of this study was to find molecular targets induced by the microenvironment by determining the in vitro and in vivo expression of proteins of the EGFR-signaling network in 6 HNSCC lines. As hypoxia is an important microenvironmental parameter associated with poor outcome in solid tumors after radiotherapy, we investigated the relationship with hypoxia in vitro and in vivo.
  
  
  METHODS
  Six human HNSCC cell lines were both cultured as cell lines (in vitro) and grown as xenograft tumors (in vivo). Expression levels were determined via western blot analysis and localization of markers was assessed via immunofluorescent staining. To determine the effect of hypoxia and pAKT-inhibition on cell survival, cells were incubated at 0.5% O(2) and treated with MK-2206.
  
  
  RESULTS
  We observed strong in vitro-in vivo correlations for EGFR, pEGFR and HER2 (rs = 0.77, p = 0.10, rs = 0.89, p = 0.03) and rs = 0.93, p = 0.02, respectively), but not for pAKT, pERK1/2 or pSTAT3 (all r(s)<0.55 and p>0.30). In vivo, pAKT expression was present in hypoxic cells and pAKT and hypoxia were significantly correlated (rs = 0.51, p = 0.04). We confirmed in vitro that hypoxia induces activation of AKT. Further, pAKT-inhibition via MK-2206 caused a significant decrease in survival in hypoxic cells (p<0.01), but not in normoxic cells.
  
  
  CONCLUSIONS
  These data suggest that (p)EGFR and HER2 expression is mostly determined by intrinsic features of the tumor cell, while the activation of downstream kinases is highly influenced by the tumor microenvironment. We show that hypoxia induces activation of AKT both in vitro and in vivo, and that hypoxic cells can be specifically targeted by pAKT-inhibition. Targeting pAKT is thus a potential way to overcome therapy resistance induced by hypoxia and improve patient outcome.
authors.affiliation: Department of Radiation Oncology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. h.stegeman@rther.umcn.nl
authors.forename: Hanneke
authors.initials: H
authors.lastname: Stegeman
authors.forename: Johannes H
authors.initials: JH
authors.lastname: Kaanders
authors.forename: Deric L
authors.initials: DL
authors.lastname: Wheeler
authors.forename: Albert J
authors.initials: AJ
authors.lastname: van der Kogel
authors.forename: Marieke M
authors.initials: MM
authors.lastname: Verheijen
authors.forename: Stijn J
authors.initials: SJ
authors.lastname: Waaijer
authors.forename: Mari
authors.initials: M
authors.lastname: Iida
authors.forename: Reidar
authors.initials: R
authors.lastname: Grénman
authors.forename: Paul N
authors.initials: PN
authors.lastname: Span
authors.forename: Johan
authors.initials: J
authors.lastname: Bussink
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003131:Combined Modality Therapy; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006258:Head and Neck Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009593:Nitroimidazoles / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D011878:Radiotherapy / Q000379:methods; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D059016:Tumor Microenvironment / Q000187:drug effects / Q000528:radiation effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D066246:ErbB Receptors; D006575:Heterocyclic Compounds, 3-Ring; D009593:Nitroimidazoles; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011838:Radiation-Sensitizing Agents; D018719:Erb-b2 Receptor Tyrosine Kinases; C548887:MK 2206; C033815:pimonidazole