title: WEE1 inhibition sensitizes osteosarcoma to radiotherapy.
abstract: BACKGROUND
  The use of radiotherapy in osteosarcoma (OS) is controversial due to its radioresistance. OS patients currently treated with radiotherapy generally are inoperable, have painful skeletal metastases, refuse surgery or have undergone an intralesional resection of the primary tumor. After irradiation-induced DNA damage, OS cells sustain a prolonged G(2) cell cycle checkpoint arrest allowing DNA repair and evasion of cell death. Inhibition of WEE1 kinase leads to abrogation of the G(2) arrest and could sensitize OS cells to irradiation induced cell death.
  
  
  METHODS
  WEE1 expression in OS was investigated by gene-expression data analysis and immunohistochemistry of tumor samples. WEE1 expression in OS cell lines and human osteoblasts was investigated by Western blot. The effect of WEE1 inhibition on the radiosensitivity of OS cells was assessed by cell viability and caspase activation analyses after combination treatment. The presence of DNA damage was visualized using immunofluorescence microscopy. Cell cycle effects were investigated by flow cytometry and WEE1 kinase regulation was analyzed by Western blot.
  
  
  RESULTS
  WEE1 expression is found in the majority of tested OS tissue samples. Small molecule drug PD0166285 inhibits WEE1 kinase activity. In the presence of WEE1-inhibitor, irradiated cells fail to repair their damaged DNA, and show higher levels of caspase activation. The inhibition of WEE1 effectively abrogates the irradiation-induced G(2) arrest in OS cells, forcing the cells into premature, catastrophic mitosis, thus enhancing cell death after irradiation treatment.
  
  
  CONCLUSIONS
  We show that PD0166285, a small molecule WEE1 kinase inhibitor, can abrogate the G(2) checkpoint in OS cells, pushing them into mitotic catastrophe and thus sensitizing OS cells to irradiation-induced cell death. This suggests that WEE1 inhibition may be a promising strategy to enhance the radiotherapy effect in patients with OS.
authors.affiliation: Department of Orthopaedic Surgery, VU University Medical Center, 1007 MB Amsterdam, the Netherlands.
authors.forename: Jantine
authors.initials: J
authors.lastname: PosthumaDeBoer
authors.forename: Thomas
authors.initials: T
authors.lastname: Würdinger
authors.forename: Harm C A
authors.initials: HC
authors.lastname: Graat
authors.forename: Victor W
authors.initials: VW
authors.lastname: van Beusechem
authors.forename: Marco N
authors.initials: MN
authors.lastname: Helder
authors.forename: Barend J
authors.initials: BJ
authors.lastname: van Royen
authors.forename: Gertjan J L
authors.initials: GJ
authors.lastname: Kaspers
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D001859:Bone Neoplasms / Q000503:physiopathology; D016203:CDC2 Protein Kinase; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D019926:Cyclin B / Q000378:metabolism; D018844:Cyclin-Dependent Kinases; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D005720:Gamma Rays; D020869:Gene Expression Profiling; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D012516:Osteosarcoma / Q000503:physiopathology; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D011743:Pyrimidines / Q000494:pharmacology; D011836:Radiation Tolerance / Q000235:genetics*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology
chemical_list: D018797:Cell Cycle Proteins; D019926:Cyclin B; D009687:Nuclear Proteins; C440869:PD 0166285; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011838:Radiation-Sensitizing Agents; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases