Blockage of epidermal growth factor receptor-phosphatidylinositol 3-kinase-AKT signaling increases radiosensitivity of K-RAS mutated human tumor cells in vitro by affecting DNA repair.
OBJECTIVE
It is known that blockage of epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K) activity enhances radiation sensitivity of human tumor cells presenting a K-RAS mutation. In the present study, we investigated whether impaired repair of DNA double-strand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RAS(mt)) cells.


METHODS
The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RAS(mt) (A549) and K-RAS(wt) (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser139), ATM (Ser1981), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr2609) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of gammaH2AX foci and frequency of micronuclei.


RESULTS
BIBX reduced clonogenic survival of K-RAS(mt)-A549 cells, but not of K-RAS(wt)-FaDu cells, after single-dose irradiation. Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RAS(mt)-A549 and MDA-MB-231 cells, but not in K-RAS(wt)-FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RAS(mt) nor in K-RAS(wt) cells the inhibitors did affect radiation-induced ATM phosphorylation. As a consequence of inhibitor treatment, a significant enhancement of both residual DSBs and frequency of micronuclei was apparent only in A549 but not in FaDu cells following radiation.


CONCLUSIONS
Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity.
Toulany
Mahmoud
M
Divison of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany.
Kasten-Pisula
Ulla
U
Brammer
Ingo
I
Wang
Shaomeng
S
Chen
Jianyong
J
Dittmann
Klaus
K
Baumann
Michael
M
Dikomey
Ekkehard
E
Rodemann
H Peter
HP
D002277:Carcinoma / Q000188:drug therapy / Q000235:genetics* / Q000532:radiotherapy; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology*; D004260:DNA Repair / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004611:Ellipticines / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D009025:Morpholines / Q000494:pharmacology*; D009154:Mutation; D009930:Organic Chemicals / Q000494:pharmacology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D011836:Radiation Tolerance / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship
C494380:9-methoxy-2-methylellipticinium acetate; C423062:BIBX 1382BS; D002867:Chromones; D004611:Ellipticines; D004791:Enzyme Inhibitors; D009025:Morpholines; D009930:Organic Chemicals; D000081082:Phosphoinositide-3 Kinase Inhibitors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt