Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair

Abstract

We have already reported that epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT signaling is an important pathway in regulating radiation sensitivity and DNA double-strand break (DNA-dsb) repair of human tumor cells. In the present study, we investigated the effect of AKT1 on DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and DNA-dsb repair in irradiated non-small cell lung cancer cell lines A549 and H460. Treatment of cells with the specific AKT pathway inhibitor API-59CJ-OH (API; 1-5 Mmol/L) reduced clonogenic survival between 40% and 85% and enhanced radiation sensitivity of both cell lines significantly. As indicated by fluorescence-activated cell sorting analysis (sub-G 1 cells) and poly(ADP-ribose) polymerase cleavage, API treatment or transfection with AKT1-small interfering RNA (siRNA) induced apoptosis of H460 but not of A549 cells. However, in either apoptosis-resistant A549 or apoptosis-sensitive H460 cells, API and/or AKT1-siRNA did not enhance poly(ADP-ribose) polymerase cleavage and apoptosis following irradiation. Pretreatment of cells with API or transfection with AKT1-siRNA strongly inhibited radiation-induced phosphorylation of DNA-PKcs at T2609 and S2056 as well as repair of DNA-dsb as measured by the ;-H2AX foci assay. Coimmunoprecipitation experiments showed a complex formation of activated AKT and DNA-PKcs, supporting the assumption that AKT plays an important regulatory role in the activation of DNA-PKcs in irradiated cells. Thus, targeting of AKT enhances radiation sensitivity of lung cancer cell lines A549 and H460 most likely through specific inhibition of DNA-PKcs-dependent DNA-dsb repair but not through enhancement of radiation-induced apoptosis.

PKB/Akt promotes DSB repair in cancer cells through upregulating Mre11 expression following ionizing radiation

“…We observed inhibition of Akt activity impaired the repair of radiation-induced DNA-DSB damage in CNE2 cells, whereas activated Akt promoted the repair of radiationinduced DNA-DSB damage in HeLa cells (Figure 1). These results were consistent with other recent studies which reported that activated Akt may decrease the efficacy of radiation therapy through promoting DNA repair in non-small-cell lung cancer cell lines and malignant-glioma cell lines (Tran et al, 2002;Kao et al, 2007;Toulany et al, 2008). Together, all these indicate that Akt can modulate radiation-induced DNA-DSB damage repair, and can regulate the efficacy of radiation therapy in other types of human cancers in which Akt kinase is hyperactivated.…”

“…The number of g-H2AX correlates with the amount of unrepaired damage (Celeste et al, 2002;Aten et al, 2004;Kao et al, 2007), and a strong correlation between g-H2AX and cell death has been reported (Banath and Olive, 2003). Thus, g-H2AX foci assay has been regarded as a sensitive and specific assay for unrepaired DSBs (Taneja et al, 2004;Bao et al, 2006;Kao et al, 2007;Toulany et al, 2008). However, this also can lead to extra DSBs when the cells progress into S phase.…”

“…One of the important reason is that activated Akt promotes survival of cells exposed to IR through inhibition of apoptosis (Brognard et al, 2001). In recent studies, it was reported that phosphoinositide 3-kinase/ Akt pathway may modulate DNA repair to affect the efficacy of radiation therapy (Tran et al, 2002;Kao et al, 2007;Toulany et al, 2008). But the precise mechanism by which Akt activation regulates repair of DNA damage remains to be determined.…”

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).…”

“…The latter protein is responsible for 'cleaning up' the DNA ends at break point, a process essential for repair initiation (Jackson, 2002). Furthermore, DNA-PK has been shown to be a co-activator of Akt (Bozulic et al, 2008), but the inverse relationship between these two kinases, with Akt being an activator of DNA-PK, has also been described (Toulany et al, 2008). This suggests that the PI3K/Akt pathway and DNA repair processes are intricately linked.…”