title: Dasatinib, a multi-kinase inhibitor increased radiation sensitivity by interfering with nuclear localization of epidermal growth factor receptor and by blocking DNA repair pathways.
abstract: OBJECTIVE
  Although inhibition of epidermal growth factor receptor (EGFR) signaling during radiation led to improvement of tumor control and survival, novel strategies are needed to further improve the outcome of patients with locally advanced head and neck carcinoma. Because EGFR is known to interact with c-Src kinases, the present study investigated dasatinib (BMS-354825), an inhibitor of c-Src kinases, for its efficacy in enhancing radiosensitivity of human head and neck squamous cell carcinomas (HNSCC) in vitro and examined the underlying mechanisms for this effect.
  
  
  METHODS
  Six HNSCC lines were exposed to dasatinib, radiation, or both, and assessed for c-Src and EGFR expression, cell survival and colony forming ability. Among these cell lines, HN-5 and FaDu lines were analyzed for induction of apoptosis, cell cycle re-distribution and for nuclear localization of EGFR, γ-H2AX and 53BP1 proteins. Immuno-precipitation and Western blots were performed to analyze the levels and binding of proteins involved in cell survival, apoptosis and DNA repair pathways. Suppression of c-Src by siRNA and subsequent clonogenic assay was performed in HN-5 cells.
  
  
  RESULTS
  All six HNSCC lines that were examined expressed high levels of c-Src. Two (HN-5 and MDA-183) expressed higher levels of EGFR than other lines. Dasatinib suppressed cell survival of all cell lines tested independent of c-Src or EGFR levels but enhanced the radiosensitivity of HN-5 and MDA-183. HN-5 and FaDu were analyzed further. Dasatinib suppressed phosphorylation of c-Src in both cell lines, but decreased repair of radiation-induced DNA damage in HN-5 cells only as evidenced by suppression of c-Abl and Nbs-1 activity, inhibition of the association between c-Src and EGFR or Her-2, prolongation of nuclear γ-H2AX and 53BP1 foci and inhibition of EGFR nuclear localization and its association with DNA-PKcs. Finally, partial suppression of c-Src resulted in a small increase in HN-5 cell radiosensitivity.
  
  
  CONCLUSIONS
  Our data demonstrate that dasatinib induces apoptosis and blocks DNA repair in EGFR-expressing HNSCC cells and improves radiotherapy outcome. These findings warrant further investigation using in vivo tumor models for potential translation into clinical testing.
authors.affiliation: University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA. uraju@mdanderson.org
authors.forename: Uma
authors.initials: U
authors.lastname: Raju
authors.forename: Oliver
authors.initials: O
authors.lastname: Riesterer
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.forename: David P
authors.initials: DP
authors.lastname: Molkentine
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Molkentine
authors.forename: Faye M
authors.initials: FM
authors.lastname: Johnson
authors.forename: Bonnie
authors.initials: B
authors.lastname: Glisson
authors.forename: Luka
authors.initials: L
authors.lastname: Milas
authors.forename: K Kian
authors.initials: KK
authors.lastname: Ang
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D017209:Apoptosis / Q000528:radiation effects; D000081247:CSK Tyrosine-Protein Kinase; D002294:Carcinoma, Squamous Cell / Q000532:radiotherapy*; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D004260:DNA Repair / Q000187:drug effects*; D000069439:Dasatinib; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006258:Head and Neck Neoplasms / Q000532:radiotherapy*; D006801:Humans; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D011836:Radiation Tolerance / Q000187:drug effects*; D000077195:Squamous Cell Carcinoma of Head and Neck; D013844:Thiazoles / Q000494:pharmacology*; D019061:src-Family Kinases / Q000032:analysis / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D066246:ErbB Receptors; D000081247:CSK Tyrosine-Protein Kinase; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; C000631346:CSK protein, human; D000069439:Dasatinib