Our past work and that of others point to increased PI3K/Akt/mTOR signalling as a mediator of enhanced tumor survival after radiation-induced DNA damage [3, 7, 9, 24]. Deregulation of mTOR signalling has also been implicated in response to radiation . Rapalogs have antiproliferative effects in vitro but their efficacy in tumors with PI3K/Akt and mTOR activation has been limited. There is extensive crosstalk between the two signalling networks [14, 17]. mTOR can affect PI3K/Akt signalling through the S6K-IRS1 feedback loop and induce Akt phosphorylation by mTORC2 [14, 25]. Because rapalogs inhibit only the mTORC1 complex, paradoxical activation of Akt can limit their therapeutic potential. Here we have shown that PI3K/mTOR dual inhibitors effectively block downstream targets and result in radiation sensitization in tumor cell lines and in endothelial cells. Interestingly, PI3K/mTOR inhibition resulted in decreased clonogenicity in cells radiated in hypoxia. These data indicate that dual PI3K/mTOR inhibition might prevent PI3K pathway reactivation and further enhance radiation-induced cell killing.
Several preclinical studies have found promising activity for the dual PI3K/mTOR inhibitor BEZ235 against various tumors [18, 19] especially those with mutations in PI3K. In the present study, dual inhibitors led to radiosensitization of tumor cells and of endothelium. The efficacy of these compounds should apply to tumor cells with a wide spectrum of oncogenic lesions because the Ras/EGFR/PI3K/mTOR pathway is activated in many types of cancer. Both BGT226 and BEZ235 enhanced the radiosensitivity of SQ20B cells and T24 cells when added before or immediately after radiation but not after 6 h. These findings may support scheduling strategies for future clinical trials testing the radiosensitising potential of these compounds.
To determine whether radiosensitisation was associated with inhibitor-mediated cell cycle redistribution, we analysed cycle distribution in cells pretreated with one of the dual inhibitors, BEZ235. Treatment of FaDu and SQ20B cells with BEZ235 alone resulted in growth arrest in the G1 phase.. This is similar to the observation reported in several studies investigating BEZ235 and other PI3K inhibitors [18, 26–28]. Importantly, when cells were irradiated after BEZ235 pretreatment, the percentage of SQ20B and FaDu cells in G2 phase was increased by approximately 3-fold and 4.5-fold, respectively. This finding concurs with our previous report on PI3K inhibitor, PI-103 where a ~2-fold increase in G2 phase population arrest was recorded . Notably, rapalogs are known to induce a G2 block when combined with irradiation .
We also investigated the effect of dual PI3K/mTOR inhibition in apoptosis. BEZ235 increased necrosis but not apoptosis in FaDu cells. In contrast, BEZ235 enhanced both apoptosis and necrosis in SQ20B cells. In the combination group, there was no increased apoptosis in either cell line and only a slight increase in necrosis was observed at 48 h post-irradiation. Previous studies have demonstrated increased apoptosis after treatment with BEZ235 in some tumor cell lines and lack of apoptosis induction in others. For instance there was no apoptosis induction in glioma or melanoma cell lines [26, 27]. There is however in lung cancer, sarcoma and leukemia [28–30].
Hypoxic cells are 2 to 3-fold more resistant than oxic cells to radiation and tumor hypoxia is associated with treatment failure following radical radiotherapy . We were therefore interested to investigate the efficacy of BEZ235 in the context of hypoxia. As expected, hypoxia resulted in increased radioresistance of FaDu, SQ20B and T24 cells. Importantly, PI3K/mTOR inhibition by BEZ235 led to significant sensitization of hypoxic cells to radiation and therefore this drug can be an attractive adjunct for radiotherapy.
BEZ235 and BGT226 enhanced persistence of residual γH2AX foci after irradiation. γH2AX foci were also moderately increased in cells treated with BEZ235 alone, which could be attributed to the potentially toxic effect of the compounds, leading to enhanced DNA damage even in the unirradiated cells. Selective inhibition of the PI3K pathway using siRNA leads to significant radiosensitization of tumor cells. Therefore, the radiosensitizing effect of PI3K/mTOR inhibitors cannot be wholly attributed to inhibition of other targets ("off-target" effect). Previous evidence has demonstrated that inhibition of the PI3K pathway can affect formation of γH2AX foci, even in the absence of radiation . These indicate that PI3K/mTOR plays a role in DNA repair after the initial injury. Our results are in accordance to the work of Konstantinidou et al. . Similar findings have been also been described before for different PI3K inhibitors [7, 31].
The PI3K/Akt/mTOR intercept node is involved in endothelial signaling response to upstream effectors such as VEGF . Chronic Akt activation in endothelial cells recapitulated the salient features of tumor vasculature . In VEGF-stimulated porcine aortic endothelial cells and HUVEC, VEGFR2 recruited the p110/p85 complex and increased their proliferation . PI3K/Akt/mTOR activation can occur upon exposure to radiation in endothelial cells . Overexpression of Akt in endothelial cells resulted in abnormal vascular remodeling with embryonic lethality . Here BEZ235 blocked VEGF- and irradiation-induced activation of Akt phosphorylation and significantly enhanced cell death in vascular and microvascular endothelial cells. Furthermore, BEZ235 reduced VEGF-mediated migration and tube formation and enhanced the antivascular effect of radiation in endothelial cells. We observed a slight increase in apoptosis and necrosis in BEZ235-treated endothelial cells. BEZ235 increased radiation-induced necrosis, especially at 24 h post-irradiation. Our findings are in accordance with previous reports showing that PI3K and/or mTOR blockade can exert an antivascular activity [15, 22, 35]. The mTOR inhibitor rapamycin decreased VEGF-mediated growth of endothelial cells and activation of Akt/mTOR signaling after irradiation and enhanced the antivascular efficacy of radiotherapy [15, 16, 22]. The fact that dual inhibition of PI3K/mTOR pathway can increase the antivascular effect of radiation in endothelial cells is an important finding. First, PI3K/mTOR inhibition by BEZ235 alone can result in alterations in tumor blood vessel morphology andfunctionality but this appears to be a dose-dependent effect and can affect the efficacy of radiotherapy significantly, as recently demonstrated by our group . Secondly, the enhanced radiosensitization of endothelial cells conferred by BEZ235 would imply that dual PI3K/mTOR inhibition could, in theory, enhance normal tissue damage and therefore special caution is required before proceeding with clinical studies using these agents in combination with radiation.