Accepted Manuscript Title: Phosphatidylinositol 3-kinase/Akt signaling as a key mediator of tumor cell responsiveness to radiation Author: Mahmoud Toulany H. Peter Rodemann PII: DOI: Reference:

S1044-579X(15)00062-0 http://dx.doi.org/doi:10.1016/j.semcancer.2015.07.003 YSCBI 1201

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Seminars in Cancer Biology

Received date: Revised date: Accepted date:

15-6-2015 9-7-2015 13-7-2015

Please cite this article as: Toulany M, Rodemann HP, Phosphatidylinositol 3-kinase/Akt signaling as a key mediator of tumor cell responsiveness to radiation, Seminars in Cancer Biology (2015), http://dx.doi.org/10.1016/j.semcancer.2015.07.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

YSCBI-D-15-00018 - Manuscript

Phosphatidylinositol 3-kinase/Akt signaling as a key mediator of tumor cell responsiveness to radiation

Mahmoud Toulany* & H. Peter Rodemann*

Division of Radiobiology and Molecular Environmental Research, Department of Radiation

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Oncology, Eberhard Karls University Tuebingen, Roentgenweg 11, 72076 Tuebingen, Ger-

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many

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*Shared corresponding author

Email: [email protected] Tel.: 07071/29-8 59 62

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Fax: 07071/29-59 00

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Prof. H. Peter Rodemann

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PD. Dr. Mahmoud Toulany

Email: [email protected] Tel: 07071/29-8 58 32

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Fax: 07071/29-59 00

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Summary The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a key cascade downstream of several protein kinases, especially membrane-bound receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) family members. Hyperactivation of the PI3K/Akt pathway is correlated with tumor development, progression, poor prognosis, and resistance to cancer therapies, such as radiotherapy, in human solid tumors. Akt/PKB (Protein Kinase B) members

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are the major kinases that act downstream of PI3K, and these are involved in a variety of cellular functions, including growth, proliferation, glucose metabolism, invasion, metastasis,

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angiogenesis and survival. Accumulating evidence indicates that activated Akt is one of the major predictive markers for solid tumor responsiveness to chemo/radiotherapy. DNA

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double-strand breaks (DNA-DSB), are the prime cause of cell death induced by ionizing radiation. Preclinical in vitro and in vivo studies have shown that constitutive activation of Akt

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and stress-induced activation of the PI3K/Akt pathway accelerate the repair of DNA-DSB and, consequently, lead to therapy resistance. Analyzing dysregulations of Akt, such as point mutations, gene amplification or overexpression, which result in the constitutive activation of

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Akt, might be of special importance in the context of radiotherapy outcomes. Such studies, as well as studies of the mechanism(s) by which activated Akt1 regulates repair of DNA-DSB,

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might help to identify combinations using the appropriate molecular targeting strategies with conventional radiotherapy to overcome radioresistance in solid tumors. In this review, we discuss the dysregulation of the components of upstream regulators of Akt as well as specific

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modifications of Akt isoforms that enhance Akt activity. Likewise, the mechanisms by which Akt interferes with repair of DNA after exposure to ionizing radiation, will be reviewed. Finally, the current status of Akt targeting in combination with radiotherapy will be discussed.

Key Words:

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PI3K/Akt, DNA repair, NHEJ, DNA-PKcs, Ionizing Radiation, Radiotherapy

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Introduction Radiotherapy, with chemotherapy and surgery, is a major cancer treatment modality used to treat approximately 50% of all cancer patients, with varying success. The dose of irradiation that can be given to a tumor is determined by the radiosensitivity of the surrounding normal tissues [1] as well as the intrinsic sensitivity/resistance of the tumor. Resistance to radiotherapy can be due either to intrinsic radioresistance or an acquired resistance during fractionated

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radiotherapy. One of the molecular events by which tumors become radioresistant is radiation-induced activation of signal transduction pathways, such as those regulated by mem-

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brane-bound receptor tyrosine kinases (RTKs) in a ligand-independent manner. In this context, the role of erbB family of receptors, especially epidermal growth factor receptor

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(EGFR), has been extensively investigated. In tumor cells, activation of EGFR stimulates signal transduction pathways that ultimately promote tumor cell proliferation, survival, migra-

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tion, invasion, and angiogenesis [2, 3]. This leads to both chemo- and radiotherapy resistance and, consequently, to a poor prognosis [4-6]. The pro-survival effect of EGFR is mediated either by nuclear accumulation of EGFR [7, 8] or by activation of various downstream signal-

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ing pathways, such as the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, the signal transducer and activator of transcription (STAT) pathway and the Ras-mitogen-activated protein

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kinase (MAPK) pathway [9-11]. The PI3K/Akt pathway is one of the major survival pathways in cancer cells, and it is frequently upregulated in human tumors [12, 13]. PI3Ks are divided into three classes according to their structural characteristics and substrate specificity

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[14]. Class I PI3Ks are further divided into class IA enzymes—which are activated by membrane-bound RTKs, G-protein-coupled receptors (GPCRs), and certain oncogenes such as the small G protein Ras—and class IB enzymes, which are regulated exclusively by GPCRs [12, 14]. Class IA PI3Ks are heterodimers that consist of a p110 catalytic subunit and a p85 regulatory subunit that, when activated, convert phosphatidylinositol-4,5-bisphosphate (PIP2) to

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phosphatidylinositol-3,4,5-triphosphate (PIP3) at the membrane, providing docking sites for signaling proteins with pleckstrin-homology (PH) domains, including phosphoinositidedependent kinase 1 (PDK1) and the Ser-Thr kinase Akt [12], . Although Akt1, when phosphorylated at the T308 residue, is active, full activation requires an additional phosphorylation at S473 by a different kinase, PDK2 [15]. So far, the kinase(s) functioning as PDK2 is/are not well described. Existing reports support both, autophosphorylation as well as phosphorylation by other kinases including ATM [16] and DNA-PKcs [17], the integrin-linked kinase 1 [18], and the mammalian target of rapamycin (mTOR)-rictor [19]. The PI3K/Akt pathway is hyperactivated in a wide range of tumor types, especially in tumors presenting a mutation in 3 Page 3 of 31

one of the components of the EGFR downstream pathways, such as phosphatase and tensin homolog (PTEN), a negative regulator of PI3K, PIK3CA and Ras [20, 21]. Mutational activation of Ras and PI3K is accompanied by resistance to radio-/chemotherapy [22-25]. Thus, the activity status of the PI3K pathway in the cytoplasm as well as in the nuclear compartment due to overexpression of RTKs or mutations in signaling components, might be a predictive marker for the responsiveness of tumor cells to radiotherapy. Akt, which is also known as

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protein kinase B (PKB), is a canonical downstream signaling effector of PI3K and an oncogene with critical roles involved in a number of important cellular processes, including cell

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growth, proliferation, survival, invasion, metastasis, and angiogenesis. [26]. In addition to these well-described functions, accumulating evidence indicates that Akt is directly involved

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in the control of DNA repair and radioresistance. In this review, the expression and activity of Akt isoforms in cancers with different origins will be summarized. We also review the role of

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Akt family members, especially Akt1, in radioresistance in solid tumors and summarize the

Role of Akt/PKB in human cancers

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role of Akt in the context of DNA double-strand break repair.

Akt/PKB is a serine/threonine kinase, which exists in three isoforms known as Akt1 (PKBα),

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Akt2 (PKBβ) and Akt3 (PKBγ). Although Akt isoforms are encoded by different genes on chromosomes 14q32, 19q13 and 1q44, respectively, their amino acid sequences share approximately 80% similarity [27]. Akt isoforms each contain three similar domains – pleckstrin

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homology, kinase and regulatory domains – and the isoforms are localized in distinct subcellular compartments [15, 28]. Akt isoforms have different functions in normal physiology and development [29]. Akt1, localized in the cytoplasm, is required for whole body normal growth [30] and mammary morphogenesis/function [31]. Akt2, co-localized with the mitochondria, is a predominant essential isoform involved in glucose metabolism, adipogenesis

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and β-cell function [32, 33]. As it is known so far, Akt3, localized in the nucleus and nuclear membrane, is essential for the attainment of normal brain size [34]. Due to the distinct subcellular localization of Akt isoforms in cancer cells [15, 28], the current model for the role of PI3K, PDK1 and PDK2 in Akt activation might only be applicable to Akt1. Over 40 downstream targets have been reported for Akt, and these contribute to the cellular roles of Akt [35]. In human malignancies, Akt activity plays a major role in tumor cell survival [36]. Increased Akt (P-S473) is associated with a lower probability of tumor control and progression free survival in cancer patients, such as those with head and neck cancers and cervical cancer after radiotherapy [37, 38]. Enhanced activation of Akt is one of the common 4 Page 4 of 31

molecular dysregulations that results from AKT gene amplification, amplificationindependent overexpression and hyperactivation of Akt through the activation of upstream pathways. [39]. Hyperactivation of Akt is a common mechanism that increases cell survival, proliferation and aggressiveness in tumors presenting overexpression of Akt upstream components, such as expression of a specific EGFR mutant (EGFRvIII, also known as EGFR type III, de2-7, and Delta EGFR) [40-42]. Mutations in RAS family members, i.e. in K-RAS and

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H-RAS, also stimulate Akt phosphorylation and tumor cell survival [43-47], mainly through upregulated production of EGFR ligands, such as amphiregulin and transforming growth fac-

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tor α. Likewise, a mutation in the PI3KCA gene that encodes P110α has been reported in many cancers to lead to enhanced concentration of PIP3 and increased Akt activity.

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Activation of PI3K and Akt are antagonized by a variety of phosphatases. PTEN, one of the most frequently inactivated tumor suppressor genes [48], is localized to the cytoplasm as well

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as the nucleus [49, 50]. PTEN acts as a lipid phosphatase in the plasma membrane that antagonizes the PI3K/Akt cell survival pathway. Point mutations, loss of heterozygosity or methylation of PTEN leads to the activation of downstream components of PI3K signaling [51, 52],

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including Akt, in tumors with different origins. Immunostaining of ovarian cancer samples revealed an inverse correlation between PTEN expression and the phosphorylation level of

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Akt [53]. Likewise, loss of PTEN is associated with a worse clinical outcome, as has been shown in patients with esophageal adenocarcinomas [54]. PTEN, in parallel with its phosphatase activity, is also involved in efficient DNA-DSB repair following genotoxic stresses such

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as exposure to ionizing radiation [49].

Previous reports have indicated that amplification, mutation, or overexpression of Akt in tumors from different origins are Akt isoform-specific phenomena [39, 55] that can result in Akt hyperactivation. Hyperactivation of Akt correlates with various clinicopathologic parameters and is a prognostic indicator for cancers from different origins, such as oral, pancrea-

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tic, thyroid and lung cancers [56-59]. Amplification of AKT1 in general is a rare phenomenon in cancers that was demonstrated for the first time in a primary human gastric adenocarcinoma [60] and later in a small population of estrogen receptor-positive breast carcinomas and prostate cancers [61]. A somatic AKT1-E17K mutation has been reported in different cancers, including breast, high-grade endometrial, bladder, lung and colorectal cancers [62-65]. Similar to Akt1, Akt2, through amplification and overexpression, also exerts oncogenic activity. For full activation, Akt2 needs to be phosphorylated at T309 and S474 [66]. Amplification of AKT2 is more common than amplification of AKT1 and has been demonstrated in human malignancies including pancreatic, colorectal, ovarian and breast cancers [67-73]. Inte5 Page 5 of 31

restingly, in ovarian cancer cells, Akt1 is phosphorylated/activated and this activation/phosphorylation is Ras-, Src- and PI3K-dependent when cells are stimulated with growth factors [74]. Mutation analyses of AKT2 revealed that somatic mutations in the AKT2 kinase domain could occur in a subset of stomach, lung, colon and breast cancers. These observations indicate that alterations in various signaling cascades that occur as a result of mutated AKT2 contribute to tumorigenesis [75]. Akt2, in association with Glut4, plays a distinct role

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in glucose transport [76]. In this context, it has been demonstrated that allosteric Akt inhibitors (SC-66 and MK-2206), which target all three Akt isoforms, decrease glucose uptake, gly-

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colysis, and cell viability in vitro [77]. This is thought to be due to the suppression of membrane localization of facilitative glucose transporter 1 (GLUT1) and its accumulation in the

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cytoplasm [78, 79]. Akt2 is also involved in autophagy of mitochondria, the so-called mitophagy process [80].

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AKT3 amplification and overexpression may play a role in the genesis of different cancers, as has been described for a subset of ovarian cancers through modulation of cell cycle progression [81] and in thyroid cancer [59], estrogen receptor-negative breast cancers, androgen-

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insensitive prostate carcinomas [82] and glioma[83]. However, so far, most studies of the carcinogenic role of Akt3 and its impact on treatment outcomes have been performed in mela-

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nomas [84, 85]. For full activity, Akt3 needs to be phosphorylated at S472 and T305 [86]. Similar to AKT1 (see above), an E17K mutation in the AKT3 gene leads to constitutive activity of Akt3 [85] and stimulation of its substrates, such as PRAS40. As reported by Madhuna-

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pantula and colleagues [87], this leads to an Akt3-dependent survival advantage and resistance to chemotherapy in melanomas [87]. Recently, Turner et al. [83], reported a specific role for amplified Akt3 in glioma progression and DNA repair pathway [83]. Akt isoforms have been identified as substrates for many different post-translational modifications that include not only the phosphorylation of the above-described residues but also

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other types of post-translational modifications, such as acetylation, glycosylation, oxidation, ubiquitination and SUMOylation [88]. These modifications might be important for additional functions of Akt isoforms in normal cells and for hyperactivation of Akt in tumor cells, even with normal levels of Akt and PTEN [89]. Understanding the network of post-translational modifications of Akt isoforms in tumor cells might help to identify novel targeting approaches for cancer therapy.

Importance of Akt in radiotherapy resistance 6 Page 6 of 31

Membrane-bound RTKs consist of 58 members that are distributed in 20 subfamilies [90]. These families of receptors are important regulators of intracellular signal-transduction pathways. Mutations and other genetic alterations result in deregulated kinase activity and the activation of downstream signaling pathways. PI3K is a crucial effector in RTK signaling, which leads to the activation of many substrates. Among those substrates activated by PI3K, Akt is one of the most important [12]. Stimulation of each member of the RTK family by re-

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lated ligands leads to the activation of the PI3K/Akt pathway. Increased basal activation of Akt in tumor cells depends on the pattern of expression of these receptors as well as on the

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presence or absence of specific mutations, such as in PTEN, PI3K, or RAS. Exposure to ionizing radiation or treatment with chemotherapy agents leads to PI3K-dependent activation of

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Akt [91]. A significant correlation exists between phosphorylated EGFR and phosphorylated Akt [92], indicating that erbB family receptors, and especially EGFR, are the major regulators

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of activation of the PI3K/Akt pathway. Overexpression or mutation of EGFR has been reported in 40 to 50 % of human solid tumors [93, 94]. Preclinical and clinical studies have demonstrated that the hyperactivation of EGFR leads to both chemo- and radiotherapy resis-

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tance and, consequently, to a poor prognosis [4, 6, 42, 81, 95-98]. Similar to ligand stimulation, exposure to ionizing radiation induces activation of EGFR and its downstream PI3K/Akt

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pathway [99-103]. PI3K/Akt activity regulates cell growth, proliferation and survival, which influences the response to ionizing radiation. The impact of PI3K/Akt activity on radioresistance has been reported by several laboratories using different cancer cell lines, including

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head and neck, colon, lung, and brain cancers in vitro [47, 99, 100, 103-105], independent of TP53 status [106], as well as in vivo [107]. Although stimulation of PI3K, in parallel with increased Akt activity, regulates the activation of other pro-survival substrates such as SGK [12], activation of Akt downstream of PI3K plays a major role in the resistance of tumor cells to different chemotherapy treatments as well as radiotherapy. In this context, Brognard and

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colleagues [108] reported that constitutive phosphorylation of Akt (presumably Akt1) at S473 and T308 mediates radiotherapy resistance in NSCLC cells in vitro [108]. In this study [108] and other studies [45, 103, 107, 109-112], radiosensitization was achieved either by pharmacological targeting of PI3K/Akt or by genetic modulation of Akt. In a similar study, Tanno et al. [113] described the expression of Akt phosphorylated at S473 in 84.2% of patients in bile duct cancer (BDC). Using BDC cell lines, these authors demonstrated that phosphorylation of Akt depends on PI3K activity [113]. Targeting PI3K with LY294002 led to a similar degree of radiosensitization, observed in cells carrying a dominant-negative Akt [113]. From these studies and the reported specific role of PI3Kβ in DNA-DSB repair [114], it can be concluded 7 Page 7 of 31

that Akt is a major mediator of PI3K-induced cellular resistance to radiotherapy. This conclusion is supported by reduced radiation sensitivity following overexpression of constitutively active Akt into tumor cells of different origins, such as NSCLC [108], bile duct cancer [113] and colon cancer [53]. PI3K/Akt activity not only leads to a limited response by tumor cells to radiotherapy but also mediates resistance to chemotherapy. This latter statement is supported by the increased cyto-

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toxicity observed in tumor cells of different origins to chemotherapy agents such as microtubule-targeting drugs (paclitaxel, docetaxel and vinblastine) [115], topotecan [116], cisplatin

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[117], and doxorubicin [118], when treatment was combined with inhibitors of components of PI3K/Akt pathway. Likewise, early evidence of antitumor activity, induced by either inhibit-

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ing Akt using MK-2206 [119] plus carboplatin and paclitaxel, docetaxel, or erlotinib, or inhibiting PI3K using the pan-class 1A PI3K inhibitor BKM120 [120] plus carboplatin and pacli-

in resistance to chemotherapy agents [119, 120].

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taxel in patients with different solid tumors, supports the above-described role for PI3K/Akt

The prognostic value of activated Akt for predicting radiation responsiveness to solid tumors

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has also been described in clinical trials. Immunohistochemical analysis of phosphorylated Akt1 at S473 implied that activated Akt1 was a potential predictive biomarker for radiothera-

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py responsiveness, in patients with head and neck cancer and cervical cancer [37, 38]. Activated Akt was also reported as a predictive marker for disease-free survival and overall survival in breast cancer patients following hormone therapy [121], and for responsiveness to

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chemotherapy with tamoxifen [122].

It has been proposed that activated Akt improves cell survival by inhibiting apoptosis as a mode of cell death. To inhibit apoptosis, Akt phosphorylates and inactivates pro-apoptotic proteins such as BAD, BAX and caspase-9. Akt-induced BAD phosphorylation prevents the binding of BAD to BCL2 family members [123]. Likewise, stimulating Akt upregulates the

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expression levels of several intracellular antiapoptotic proteins, such as FLIP, survivin, cIAP1, cIAP-2, A1/Bfl-1, and XIAPs [124, 125]. Akt-mediated inhibition of apoptosis abrogates the sensitivity of hematopoietic cells, including leukemia cells, to therapeutic approaches that induce apoptosis [125]. Ionizing radiation can also induce apoptosis through a mitochondria-dependent intrinsic pathway [126]. In this pathway, activated Akt phosphorylates the proapoptotic proteins Bax and Bad. Phosphorylation of Bax at S184, which is regulated in an Akt-dependent manner, inhibits the effects of Bax on mitochondria by maintaining the protein in the cytoplasm [127]. Yet, heterogeneous effects of Akt inhibition on radiationinduced apoptosis have been reported. We showed that targeting Akt results in radiation8 Page 8 of 31

induced apoptosis in neither apoptosis-sensitive nor apoptosis-resistant NSCLC cell lines [128]. In both cell types, Akt targeting led to an inhibition of repair of radiation-induced DNA-DSB and the subsequent enhancement of radiation sensitivity [128]. In line with this observation, Hambardzumyan et al. [129] demonstrated that inhibition of Akt in malignant gliomas mediated radiosensitization independent of apoptosis [129]. In the majority of previous reports that have investigated the role of apoptosis on post-irradiation cell survival fol-

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lowing Akt inhibition, increased levels of apoptosis or prerequisite biochemical changes, such as caspase cleavage [130], have been interpreted as a mechanism of radiosensitization. For

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example, activated Akt in human bile duct cancer is associated with increased radioresistance, and its targeting leads to increased apoptosis and radiosensitization [113]. In a study by Tanno

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et al. [113], the role of increased apoptosis after Akt targeting was explored as a mechanism of radiosensitization. This argument is also applicable to those studies, in which inhibition of

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Akt and increased apoptosis were observed after targeting of upstream regulators of Akt, such as EGFR [131]. Given that radiosensitization can be achieved by enhancing different modes of cell death , such as mitotic catastrophe, autophagy, senescence [132-134] and apoptosis,

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the specific role of apoptosis as a mechanism of radiosensitization in solid tumors in general, and especially following Akt targeting, is questionable and requires further investigation.

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Evidence from experimental and clinical studies indicates that cancer-initiating cells, or cancer stem cells (CSC), are resistant to radiotherapy [135]. In this context, CD44 expression has the potential to predict the outcome of radiotherapy assessed by CSC density [135]. Enhanced

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CSC phenotypes are associated with the activation of the PI3K/Akt/mTOR pathway in radioresistant prostate cancer [136]. Moreover, it has been demonstrated that the expression of Akt1 and Akt2 increased after irradiation in MCF-7 mammosphere CD24(-/low)/CD44(+) expressing cells, but not in the bulk population of MCF-7 CD24(+)/CD44(+) expressing cells [137]. In this study [137], targeting of Akt sensitized MCF-7 mammosphere cells, but not

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MCF-7 monolayer cells to ionizing radiation. Thus, it seems that Akt isoforms, through an as yet unknown mechanism, are involved in radioresistance in CSCs. In this context, varying effects of Akt isoforms on the expression of the cancer stem cell markers CD133 and CD44 [138], in association with radioresistance in tumors of different origin [139, 140], have been reported. The role of Akt activity in radioresistance in CSCs has been demonstrated in both mammospheres in vitro and in vivo [141]. Zhang et al. [141] reported that inhibition of the Akt pathway selectively inhibited canonical Wnt signaling as well as repair of DNA damage in cancer initiating cells and sensitized them to ionizing radiation in vitro and in vivo [141]. At least for Akt1, it has been described that it phosphorylates stem cell marker Oct4 at threo9 Page 9 of 31

nine 235 in embryonal carcinoma cells [142]. Phosphorylated Oct4 increases its stability and facilitated Oct4 nuclear localization. In the nucleus, Oct4 interacts with Sox2, which promotes the transcription of the core stemness genes POU5F1 and NANOG [142]. With respect to the described functional role of the PI3K/Akt pathway in CSCs [129, 143] and the role of cancer stem cells in radiotherapy outcomes [135], inhibition of the Akt pathway might offer an improved response to radiation in CSCs [137, 141, 144]. Based on the assumption that CSCs are

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more radioresistant and that they present Akt pathway hyperactivation, our laboratory has shown that a selected radioresistant subpopulations of NSCLC-A549 cells that present the

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CSC marker ALDH1 can be radiosensitized by the PI3K inhibitor LY294002 [145]. The importance of PI3K/Akt signaling to radioresistance in CSCs is also underlined by data indicat-

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ing the Akt-dependence of accelerated repair of radiation-induced DNA-DSB [141], especially in brain tumor stem cell [146]. Thus, regulation of tumor cell stemness might be one of the

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mechanisms by which Akt can promote the survival and tumorigenicity

In recent years, autophagy has been recognized as an important process in carcinogenesis as well as in processes mediating the responses of tumor cells to therapy, especially radiation

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therapy [147-149]. Because inhibition of autophagy, through either autophagy inhibitors or genetic approaches, induces radiosensitization in vitro [147, 149], and autophagy inhibitor

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chloroquine may improves radiotherapy outcome [150], autophagy induction by irradiation is most likely a friend rather than a foe for cancer cells. Evidence shows that the prosurvival functions activated by the PI3K/Akt pathway are in part due to the stimulation of autophagy

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[133, 151]. However, no convincing data exists supporting Akt-dependent, autophagymediated cell survival after exposure to ionizing radiation. A report by Fujiwara et al. [152] demonstrated that targeting Akt with an Akt inhibitor mediated radiosensitization in glioma cells by inducing autophagy. In this study, the specific inhibition of Akt using the DN-Akt plasmid radiosensitized U87 cells expressing a constitutively active form of epidermal growth

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factor receptor (U87-MGΔEGFR) by enhancing autophagy [152]. Thus, further studies are needed to identify the mechanism(s) involved in the cytoprotective effects of radiationinduced autophagy and the cytotoxic effects of Akt-induced autophagy on post-irradiation survival. Although, compared to solid tumors, hematological malignancies including leukemia, myeloma and lymphoma are very sensitive to ionizing radiation, radiotherapy is not the standard first-line treatment approach against these cancers and is used only in specific circumstances. Instead, chemotherapy, immunotherapy and stem cell transplantation are the major treatment modalities. In addition, rationally designed targeted therapies such as small molecule tyrosine 10 Page 10 of 31

kinas inhibitors (TKI), antibodies, antibody-cytotoxic agent combinations are going to be established as disease specific standard approaches. For instance, effect of imatinib as BCRABL TKI has become promising in chronic myeloid leukemia [153]. Likewise, application of monoclonal antibody rituximab that targets CD20 protein, expressed in mature B-cells, is used as single agent therapy or in combination with chemotherapy to target some types of lymphomas [154]. Similar to solid tumors, PI3K/AKT pathways is frequently hyperactivated

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in hematological malignancies such as in childhood acute lymphoblastic leukemia, acute myelogenous leukemia, and chronic myelogenous leukemia, as well as in some pediatric lym-

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phomas and lymphoproliferative disorders (22845486). This is mainly due to mutations in the upstream regulator of the pathway such as Fms-like tyrosine kinase 3 (FLT3) [155] and N-

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RAS [156, 157] as well as overexpression of IGF-1 receptor [158] or constitutive activation of IGF-1 receptor through autocrine secretion of related ligands.

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Thus, based on the experience with imatinib, single targeting of the anti-apoptotic PI3K/Akt pathway or the combination of PI3K/Akt inhibitors with chemotherapy agents or other rationally designed molecular approaches might be effective strategy to control hematological ma-

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lignancies. For example, perifosine that inhibits Akt activity has cytotoxic activity against multiple myeloma cell lines, and it enhances the cytotoxic effects of drugs such as

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doxorubicin and dexamethasone by enhancing apoptosis [159]. Analyzing apoptosis in hematological malignancies is a valuable approach to evaluate treatment efficacy, e.g. after targeting PI3K/Akt pathway. The status of targetability of the PI3K/Akt pathway in hemato-

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logical malignancies has also been well reviewed by other authors [160-162]

Control of DNA damage by Akt as an important mechanism for radioresistance Previous reports showed a direct EGFR-Akt correlation, indicating that EGFR was a major regulator of Akt-dependent DNA-DSB repair [97, 163-165] after irradiation. Moreover, the

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impact of Akt activity on DNA-DSB repair and radioresistance in tumor cells from different origins has also been demonstrated [23, 102, 128, 166, 167]. DNA-DSB are the most lethal type of DNA lesions that lead to cell death following exposure to ionizing radiation [168]. The two pathways involved in DNA-DSB repair are non-homologous end-joining (NHEJ) and homologous recombination (HR) [169], but NHEJ is the predominant pathway in mammals. The DNA-dependent protein kinase catalytic subunit complex (DNA-PKcs) is a major enzyme in the NHEJ process and response to radiotherapy [170, 171]. Phosphorylation of DNA-PKcs at specific amino acid residues, including the T2609 cluster and S2056, are required and essential for efficient repair of DNA-DSB during NHEJ [172]. Enhanced cellular 11 Page 11 of 31

sensitivity to irradiation after mutations at these phosphorylation sites supports the specific function of DNA-PKcs phosphorylation in DNA-DSB repair [173-175]. Previous reports from our laboratory [128, 166] and a report by Park and colleagues [176] demonstrated that Akt directly interacts with DNA-PKcs through its C-terminal domain. Akt1 and DNA-PKcs form a functional complex immediately after irradiation in the nucleus exposure [166, 177]. Using GFP-tagged DNA-PKcs expressing cells, we were able to demonstrate for the first time

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that Akt and especially Akt1 promotes DNA-PKcs accumulation at the DNA-DSB site and stimulates DNA-PKcs kinase activity, which is a necessary step for progression of DNA-DSB

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repair [166]. Obtained data by using the Akt inhibitors indicates that Akt activity regulates ATM mediated transphosphorylation of DNA-PKcs at T2609 as well [111, 163]. This obser-

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vation is further supported by the role of Akt1 isoform in T2609 phosphorylation in lung cancer cells [83] and the role of Akt3 in ATM-dependent DNA-PKcs phosphorylation in gliomas

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after exposure to ionizing radiation. Akt1-dependent DNA-PKcs activity/phosphorylation is essential for efficient repair [175] as well as the release of DNA-PKcs from the damage site. The role of Akt in DNA-DSB repair is further substantiated by the co-localization γH2AX

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foci with P-Akt after irradiation [166, 167, 177, 178]. Based on these results, a detailed mechanism for activation of DNA-PKcs by Akt (summarized in Fig. 1) can be proposed. Akt,

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activated by mutation or overexpression of upstream components such as erbB receptors [41, 102, 163, 179], PTEN [23, 180], Ras [46, 111, 181] and PI3K [163], results in increased radiation-induced DNA-PKcs activity and the enhanced repair of DNA-DSB. Thus, in the case of

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dysregulation of these molecules, a direct targeting of Akt, but not upstream components might be an efficient approach for radiosensitization. Based on this argument, it is proposed that the targeting of PI3K in combination with radiotherapy leads to heterogeneous responses that depend on the AKT genotype, i.e., radiosensitization in AKT wild-type tumor cells and a lack of radiosensitization in Akt-mutated tumor cells.

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An alternative pathway by which Akt regulates DNA-DSB repair is the upregulation of MRE11 expression after Akt activation through the Akt/GSK3β/β-catenin/LEF-1 pathway [171]. MRE11 is a central protein that binds to RAD50 and NBS1. MRE11, RAD50 and NBS1 (MRN) complex, after irradiation, rapidly accumulates to damage sites and remains there until the damage has been repaired. The MRN complex appears to be the major sensor of these breaks, and it subsequently recruits ATM, which is in turn activated to phosphorylate members of the MRN complex and a variety of other proteins that are involved in cell-cycle control and DNA repair [182]. Approximately 85% of DNA-DSB induced by ionizing radiation are repaired within the first 2-3 h post-irradiation by the fast component of DNA-DSB 12 Page 12 of 31

repair, which has been shown to be independent of ATM function [183]. The remaining 15% of DNA-DSB, which mainly include complex lesions, are repaired in an ATM-dependent manner by the slow component [184, 185]. Because targeting Akt leads to the downregulation of MRE11 at the transcriptional level, the role of Akt1 in DNA repair under this mechanism might be due to regulation of slow components of DNA repair, a complementary mechanism to the Akt/DNA-PKcs-dependent fast repair process. As reported by Viniegra et al. [186], full

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activation of Akt in response to insulin and ionizing radiation is ATM-dependent. In this context, Fraser and colleagues [167] showed that the activation of the MRE11-ATM-RNF168

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pathway induced Akt phosphorylation and that this led to Akt-dependent enhanced repair of DNA-DSB [167]. In line with the role of ATM in Akt activity after irradiation [187], it has

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been reported that ATM-dependent Akt signaling regulated DNA-DSB repair in cells exposed to clinically relevant doses of irradiation [188, 189]. As summarized in Fig. 1, because of

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ATM-mediated transphosphorylation of DNA-PKcs at T2609 [175] as well as the role of Akt in T2609 phosphorylation [83, 111, 128], activation of Akt/DNA-PKcs in the nucleus is at least partially ATM-dependent (Fig. 1). Akt-dependent activation and the phosphorylation of

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DNA-PKcs [111, 128, 166] indicate that Akt is involved in the fast component of DNA-DSB repair. Likewise, because ATM phosphorylates Akt [187] and Akt activity upregulates

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MRE11 [171], which is a slow process, it can be concluded that Akt is also part of the slow component of DNA-DSB repair. These two aspects need to be investigated further. In contrast to NHEJ, which in general is active throughout the cell cycle and does not rely on

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a template, HR is restricted to the S- and G2-phases of the cell cycle due to the requirement of the presence of a sister chromatid as a template. BRCA1, BRCA2 and RAD51 are the major proteins that regulate HR repair. As described for hereditary breast and ovarian cancers, BRCA1 and BRCA2 are frequently mutated in these cancers [190, 191], possibly due to genomic instability as a consequence of HR deficiency. A link between Akt1 activity and HR

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has also been demonstrated. HR deficiency is associated with increased phosphorylation of Akt1 at S743, as shown in breast cancer patients. Likewise, tumor formation due to BRCA1 deficiency is reduced by Akt1 depletion [192]. Inhibition of HR by Akt1 activity in BRCA1proficient breast cancer cells has been demonstrated to be due to the induction of cytoplasmic retention of BRCA1 and RAD51 [193]. In HR-deficient cells, the Akt1-mediated inhibition of HR occurs through impaired Chk1 nuclear localization and the subsequently disruption of Chk1-Rad51 interactions [194]. In contrast to this observation, Akt3 overexpression in high grade glioma war reported to enhance HR [83]. Thus it seems that aberrant activation of Akt may differentially affect HR in different tumor cells [193]. 13 Page 13 of 31

PTEN activity leads to inactivation of PI3K/Akt pathway. Regarding the role of PTENregulated Akt activity in DNA-DSB repair, conflicting reports exist. Puc et al. [195] demonstrated that PTEN knockdown generated DNA damage due to insufficient inactivation of CHK1 under non-irradiated basal conditions. In this report [195], although a γH2AX foci assay in control-siRNA transfected and irradiated cells was used as a positive control, the effect of PTEN-siRNA on residual DNA-DSB was not investigated. Thus, this study did not provide

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strong evidence that inhibition of HR is mediated by Akt activity. Moreover, a link between nuclear PTEN and HR repair has been reported, in which SUMOylation of nuclear PTEN [49]

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as well as PTEN interaction with Rad52 [196] were described to be required for HR [49, 196]. Since it is not clear whether activation of nuclear Akt by PDK1 is similar to that in the cyto-

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plasm, role of Akt in HR regulated by nuclear PTEN cannot be currently demonstrated. Further study by Kao et al. [23] demonstrated that overexpression of PTEN resulted in decreased

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Akt1 phosphorylation and repair of DNA-DSB. This study does not support a role for increased Akt activity in hampering HR, as has been suggested by others [192-194]. However, if Akt activity leads to inhibiting HR, because DNA-DSB repair involves both the NHEJ and

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HR repair pathways, the negative effect of Akt on HR might be dominated by Akt-mediated stimulation of NHEJ. Under this condition Akt targeting will finally lead to enhance residual

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DSB, leading to increased radiation response. Additionally, a study by Li et al. [197] indicated that the EGFR tyrosine kinase inhibitor erlotinib attenuated HR repair of chromosomal breaks in human breast cancer cells after irradiation [197]. Because erlotinib inhibits Akt

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phosphorylation, from this study and a study by Kao et al. [23] can be concluded that Akt has a differential effect on HR in tumor cells after irradiation compared to non-irradiated conditions. Nevertheless, the distinct role of Akt in repair of DNA-DSB through the HR pathway needs to be further investigated.

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Akt inhibitors in cancer therapy Extensive crosstalk at different levels between the PI3K/Akt pathway and the MAPK/ERK pathway is an obstacle to single-targeting PI3K in tumors, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity [198]. In addition to the previously described crosstalk between these two pathways, we identified a novel crosstalk mechanism by which 24 h inhibition of PI3K by PI-103 [199] or LY294002 (Toulany and Rodemann, unpublished data) resulted in the MAPK-dependent reactivation of Akt in tumor cells by constitutive K-Ras activity. Reactivated Akt diminished the anti-clonogenic effect of PI-103 [199] and its radiosensitization when combined with therapeutic doses of ionizing rad14 Page 14 of 31

iation (Toulany and Rodemann, unpublished data). Targeting PI3K using the previously described PI3K inhibitors, such as LY294002, results in the broad-spectrum inhibition of multiple signaling pathways and induces toxicity in normal tissues. Thus, based on the toxicity of PI3K inhibitors and the described crosstalk between PI3K/Akt and MAPK/ERK pathway [200], in addition to the compensatory activation of Akt by PI3K inhibition [199], we suggest that specifically targeting Akt might be an alternative and more effective approach for anti-

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cancer therapeutics than using PI3K inhibitors.

Akt inhibitors are either ATP mimetic kinase inhibitors or allosteric inhibitors that bind to the

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pleckstrin-homology domain of Akt, which have been described to target all the 3 Akt isoforms. Current clinical oncology trials are in different phases of testing the feasibility and

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applicability of these inhibitors. In general, obtained data from allosteric inhibitors such as perifosine in different advance neoplasms [201, 202] and nelfinavir in glioblastoma and local-

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ly advanced rectal cancers [203, 204] indicate a favorable safety profile. Akt allosteric inhibitor MK2206 in combination with cytotoxic therapies [119, 205] or in combination with other molecular targeting agents such as anti-IGF-1R antibody dalotuzumab [206] and EGFR ki-

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nase inhibitor erlotinib [119] was shown to be well-tolerated in phase I trials. For hematologic malignancies, a favorable safety profile and clinical activity against multiple myeloma has

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been demonstrated by Akt allosteric inhibitor afuresertib alone in phase I study (GSK2110183)[207]. Likewise, in a phase I/II study, perifosine-bortezomib ± dexamethasone demonstrated encouraging activity in heavily pretreated bortezomib-exposed patients with

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advanced multiple myeloma [201]. Antisense strategy has also been employed to develop Akt inhibition. RX-0201 is an antisense oligonucleotide to Akt1, which shows growth inhibition of various cancer cells at nano molar concentrations [208]. Altogether, data obtained from these studies and several other studies reviewed elsewhere [208-211] demonstrate the potential benefits of Akt inhibitors in clinical use. Thus, according to the results of phase I/II clini-

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cal trials of Akt inhibitors and the preclinical as well as clinical reports supporting the role of Akt in radioresistance, Akt targeting strategies, and especially targeting of Akt1, prior to radiotherapy might be a highly effective strategy to overcome PI3K/Akt-dependent radioresistance in solid tumors. To this aim, as summarized in table 1, so far only few phase I clinical investigations have completed (Table 1) following combining perifosine or nelfinavir with radiotherapy. Pharmacokinetic study of combined treatment with perifosine and radiotherapy has been performed in patients with advanced solid tumors including NSCLC, prostate, esophageal, colon and bladder cancer [212]. According to this study, perifosine can be safely combined with fractionated radiotherapy at a dosage of 150 mg/day, to be started at least 1 15 Page 15 of 31

week prior to radiotherapy. This study has been recommended for phase II evaluation [212]. Likewise, nefinavir, administered concurrently with chemoradiotherapy, had acceptable toxicity in patients with NSCLC [213], glioblastoma [203], rectal carcinoma [204] and pancreatic cancer [214]. Importantly, the first tumor response evaluations was promising following combination of nelfinavir and chemoradiotherapy in locally advanced rectal cancer [204] and NSCLC [213]. Further studies are needed to get more knowledge for the toxicity as well as

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the efficacy of this treatment regimen in different tumors presenting hyperactivation of

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PI3K/Akt pathway.

Conclusion

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Understanding the function of cellular signaling pathways involved in tumor growth, proliferation and survival is important for designing molecular targeting approaches in oncology.

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Activation of the PI3K/Akt signaling pathway is crucial for post-irradiation cell survival. Most of the small-molecule inhibitors used to target signal components within this pathway are cytostatic rather than cytotoxic. Likewise, hyperactivation of the downstream components

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of this pathway, such as mutations in PTEN, PI3K, or AKT results in a lack of response to inhibitors of upstream regulators. Although erbB family members are the major regulators of

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the PI3K/Akt pathway, this pathway can also be regulated by many other receptors, such as GPCRs, integrin receptors and insulin receptors. Thus, direct targeting Akt not only diminishes the crosstalk between the pathways but it can also shift the cytostatic effect of Akt inhibi-

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tors towards an increased radiotoxicity by inhibiting DNA-DSB. Thus, Akt rather than RTKs or PI3K should be the prime molecule to be targeted to overcome radiotherapy resistance in solid tumors that present enhanced PI3K/Akt signaling.

Conflict of interest

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The authors declare that there are no conflicts of interest.

Acknowledgement Supported by grants from the Deutsche Forschungsgemeinschaft [Ro527/7-1 and SFB-773TP B02] awarded to HPR, GRK 1302/2 (T11) awarded to MT/HPR.

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Fig. 1: A scheme illustrating the potential role of a PI3K/Akt-mediated pathway in radiation responses After irradiation, activated Akt improves post-irradiation cell survival through several mechanisms. Akt translocates from the cytoplasm to the nucleus. In the nucleus Akt1 can also be activated by the MRE11-ATM pathway [167]. Nuclear Akt1 interacts with and forms a functional complex with DNA-PKcs. In this complex, Akt1 stimulates DNA-PKcs kinase activity,

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DNA-PKcs autophosphorylation and DNA-PKcs accumulation at DNA-double strand breaks.

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This leads to the efficient repair of DNA-DSB and, consequently, to radioresistance.

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29 Page 29 of 31

YSCBI-D-15-00018 - Table 1

Combination with chemotherapy

Cancer type

Clinical trial phase

Observed toxicity

Perifosine

No

Phase I Ref. 212

Acceptable

Nelfinavir

Temozolamide

NSCLC, Prostate, Oesophageal, Colon and Bladder cancer GBM

Phase I Ref. 203

Nelfinavir

Capecitabine

Rectal

Nelfinavir

Cisplatinum and Etoposide

NSCLC

Nelfinavir

Cisplatin

Pancreatic

Phase I Ref. 204 Phase I Ref. 213 Phase I Ref. 214

Acceptable, further monitoring of hepatotoxicity needed for higher dosage. Stage III Stage III

Stage III/IV

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Agent

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Table 1: Summary of existing phase I clinical trials of Akt allosteric inhibitors perifosine and nelfinavir in combination with chemoradiotherapy.

Page 30 of 31

erbB1

EGFR

erbB2 erbB2

us

EGFR

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YSCBI-D-15-00018 - Figure 1

Stemness

PDK1

PDK2

Akt

Metabolism

Autophagy

ed

Apoptosis

ce pt

Akt

ATM

erbB2

M an

Fig. 1

Akt

Akt

DNA-PKcs

Ac

DNA-PKcs

MRN

IR-induced DNA-DSB

Akt DNA-PKcs

XLF XRCC4

Akt DNA-PKcs

Ligase IV

KU70/80

KU70/80

DNA-DSB repair Page 31 of 31