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

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 doublestrand 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 (Ser 139 ), ATM (Ser 1981 ), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr 2609 ) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of gH2AX 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 Thr 2609 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.

The pyridinylfuranopyrimidine inhibitor, PI-103, chemosensitizes glioblastoma cells for apoptosis by inhibiting DNA repair

“…In tumor cells, however, which are usually more dependent on PI3K-mediated survival signaling and frequently express stimuli-independent activity of this signaling cascade (Yuan and Cantley, 2008), the relationship between PI3K and DNA-PK can be altered. Here, Akt has been reported to be an activator of DNA-PK, responsible for its phosphorylation (Toulany et al, 2006(Toulany et al, , 2008. These findings are consistent with our own data, which show that in doxorubicin-treated GBM cells, Akt is not downstream of DNA-PK, as inhibition of the latter does not affect phosphorylation of the former (Supplementary Figure 3d).…”

Caffeine Confers Radiosensitization of PTEN-Deficient Malignant Glioma Cells by Enhancing Ionizing Radiation–Induced G1 Arrest and Negatively Regulating Akt Phosphorylation

“…It has been shown by numerous studies that the activation of the PI3K/Akt signaling pathway in tumor cells, either by increased growth factor receptor signaling (20,21) or decreased activity of specific pathway inhibitors such as Figure 4. The interaction of caffeine with p53/p21 Waf1 and p-Akt Ser437 expression levels.…”

Stimulated PI3K-AKT Signaling Mediated through Ligand or Radiation-Induced EGFR Depends Indirectly, but not Directly, on Constitutive K-Ras Activity

“…AKT kinase is efficiently induced by growth factors like EGFR ligands or by ionizing radiation through the EGFR-mediated activation of PI3K (28)(29)(30)(31). PI3K itself is a heterodimeric complex composed of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit.…”

“…It seems to be clear that activation of AKT via PI3K occurs through phosphorylation by phosphoinositide-dependent kinase-1 and/or phosphoinositide-dependent kinase-2 at serine and threonine residues (Ser 472/473 , Thr 308 ). Several authors reported that especially phosphorylation of AKT at Ser 473 is associated with resistance to chemotherapy/ radiotherapy (36)(37)(38)(39)(40), and it has been proposed that activated AKT promotes survival of cells exposed to ionizing radiation through inhibition of apoptosis (41,42) or enhancement of DNA double-strand break repair (31).…”