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 γH2AX 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.

More than half of cancer patients receive radiotherapy as a part of their cancer treatment. DNA double-strand breaks (DSBs) are considered as the most lethal form of DNA damage and a primary cause of cell death and are induced by ionizing radiation (IR) during radiotherapy. Many malignant cells carry multiple genetic and epigenetic aberrations that may interfere with essential DSB repair pathways. Additionally, exposure to IR induces the activation of a multicomponent signal transduction network known as DNA damage response (DDR). DDR initiates cell cycle checkpoints and induces DSB repair in the nucleus by non-homologous end joining (NHEJ) or homologous recombination (HR). The canonical DSB repair pathways function in both normal and tumor cells. Thus, normal-tissue toxicity may limit the targeting of the components of these two pathways as a therapeutic approach in combination with radiotherapy. The DSB repair pathways are also stimulated through cytoplasmic signaling pathways. These signaling cascades are often upregulated in tumor cells harboring mutations or the overexpression of certain cellular oncogenes, e.g., receptor tyrosine kinases, PIK3CA and RAS. Targeting such cytoplasmic signaling pathways seems to be a more specific approach to blocking DSB repair in tumor cells. In this review, a brief overview of cytoplasmic signaling pathways that have been reported to stimulate DSB repair is provided. The state of the art of targeting these pathways will be discussed. A greater understanding of the underlying signaling pathways involved in DSB repair may provide valuable insights that will help to design new strategies to improve treatment outcomes in combination with radiotherapy.

Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation.

Akt1 is known to promote non-homologous end-joining (NHEJ)-mediated DNA double-strand break (DSB) repair by stimulation of DNA-PKcs. In the present study, we investigated the effect of Akt1 on homologous recombination (HR)-dependent repair of radiation-induced DSBs in non-small cell lung cancer (NSCLC) cells A549 and H460. Akt1-knockdown (Akt1-KD) significantly reduced Rad51 protein level, Rad51 foci formation and its colocalization with γH2AX foci after irradiation. Moreover, Akt1-KD decreased clonogenicity after treatment with Mitomycin C and HR repair, as tested by an HR-reporter assay. Double knockdown of Akt1 and Rad51 did not lead to a further decrease in HR compared to the single knockdown of Rad51. Consequently, Akt1-KD significantly increased the number of residual DSBs after irradiation partially independent of the kinase activity of DNA-PKcs. Likewise, the number of residual BRCA1 foci, indicating unsuccessful HR events, also significantly increased in the irradiated cells after Akt1-KD. Together, the results of the study indicate that Akt1 seems to be a regulatory component in the HR repair of DSBs in a Rad51-dependent manner. Thus, based on this novel role of Akt1 in HR and the previously described role of Akt1 in NHEJ, we propose that targeting Akt1 could be an effective approach to selectively improve the killing of tumor cells by DSB-inducing cytotoxic agents, such as ionizing radiation.