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

Clin Cancer Res. 2006 Jul 1;12(13):4119-26. doi: 10.1158/1078-0432.CCR-05-2454.

Abstract

Purpose: 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.

Experimental design: 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma / drug therapy
  • Carcinoma / genetics*
  • Carcinoma / radiotherapy
  • Cell Line, Tumor
  • Chromones / pharmacology*
  • DNA Repair / drug effects*
  • Dose-Response Relationship, Drug
  • Ellipticines / pharmacology
  • Enzyme Inhibitors / pharmacology
  • ErbB Receptors / antagonists & inhibitors*
  • Genes, ras / genetics*
  • Humans
  • Morpholines / pharmacology*
  • Mutation
  • Organic Chemicals / pharmacology*
  • Phosphoinositide-3 Kinase Inhibitors*
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors
  • Radiation Tolerance / drug effects
  • Signal Transduction / drug effects
  • Structure-Activity Relationship

Substances

  • 9-methoxy-2-methylellipticinium acetate
  • BIBX 1382BS
  • Chromones
  • Ellipticines
  • Enzyme Inhibitors
  • Morpholines
  • Organic Chemicals
  • Phosphoinositide-3 Kinase Inhibitors
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • ErbB Receptors
  • Proto-Oncogene Proteins c-akt