AKT Is Activated in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner in Response to Temozolomide and Confers Protection against Drug-Induced Cell Growth Inhibition

Abstract

The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)-and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinasedead form of AKT1 into breast cancer cells abrogated TMZinduced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor D-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)-propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)-mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O 6 -guanine-methylating agents.The serine/threonine protein kinase AKT is a critical regulator of major cellular processes, including gene expression, glycogen metabolism, migration, proliferation, and survival (for review, see Bellacosa et al., 2005). Three isoforms of AKT, encoded by three different genes, are present in mammalian cells. Each isoform comprises an NH 2 -terminal pleckstrin homology domain, a catalytic protein kinase domain, and a short COOH-terminal region. All AKT isoforms contain two main regulatory phosphorylation sites, one within the kinase domain (Thr308/AKT1, Thr309/AKT2, Thr305/ AKT3), and one in the COOH-terminal region (Ser473/AKT1, Ser474/AKT2, Ser472/AKT3).This study was supported by the Italian Ministry of Health. Article, publication date, and citation information can be found at

LY294002 enhances cytotoxicity of temozolomide in glioma by down-regulation of the PI3K/Akt pathway

“…Nagane et al (22) and Weller et al (23) showed that up-regulation of either Bcl-2 or EGFR expression in glioma cell lines was correlated with drug resistance and decreased apoptotic response in vitro. Caporali et al demonstrated that Akt was activated in an ataxia telangiectasia and Rad3 related-dependent manner in response to TMZ and conferred protection against druginduced cell growth inhibition (24). These results strongly support the hypothesis that clinical benefits are obtained by combining TMZ with inhibitors of the Akt pathway.…”

DNA-dependent Protein Kinase-mediated Phosphorylation of Protein Kinase B Requires a Specific Recognition Sequence in the C-terminal Hydrophobic Motif

“…In addition, mTOR and its associated protein Rictor have been shown to phosphorylate PKB on Ser-473 in Drosophila and human cells (14,46). Recently, it has also been reported that ATR, another member of the PI3K-like kinase, may act as a novel upstream activator of PKB in response to DNA damage induced by O 6 -guanine methylating agents (47). These data suggest that phosphorylation of PKB at Ser-473 is a complex and important regulatory process in cells and further underscore the key role of PKB activation in many physiological processes.…”

Cancer stem cells and escape from drug-induced premature senescence in human lung tumor cells: Implications for drug resistance and in vitro drug screening models

“…35,36 Importantly, Akt/PKB kinase is one of the targets of both ATM and ATR kinases as well as DNA-PK. [37][38][39] Caffeine has also been shown to inhibit mTOR kinase in vitro at low milimolar concentrations. 30 Moreover, recent reports showed that cellular senescence induced in immortalized epithelial cells by overexpression of p16 and p21, two inhibitors of cyclin-dependent kinases, may be decelerated by a specific mTOR inhibitor rapamycin.…”