Figure 3 Review flow diagram of the publication selection in preclinical category.
1Exclusion criteria include the retracted publication, duplicate publication or non-original papers, including review articles, letters and editorials, comments, case reports, etc.; 2According to the scope of the current review: Efficacy of Akt modulation by 9 DNA-targeted agents in 5 types of cancer, including lung cancer, malignant mesothelioma, pancreatic cancer, ovarian cancer, and malignant glioma. PI3K: Phosphatidylinositol 3 kinase; EGFR: Epidermal growth factor receptor; mTOR: Mammalian target of rapamycin.
Effect of Akt-inhibition on taxane sensitivity
LY294002, Wortmannin, BEZ235, or perifosine-mediated inhibition of the PI3K/Akt-dependent survival pathway enhanced paclitaxel cytotoxicity in various cancers, e.g., malignant glioma[63,80], lung[50,64-66,80], esophageal[64,80], and ovarian cancer cells[32,56,61,67-70,88] (Table 3). However, there are some data not in favor of the combination. LY294002 did not potentiate cisplatin, pemetrexed, or paclitaxel in A549 lung adenocarcinoma cells harboring K-ras mutation and wild-type EGFR. Likewise, inactivation of PI3K/Akt signaling by LY294002 did not result in significant alteration of sensitivity of human ovarian carcinoma A2780 cells to paclitaxel. Similarly, the combination of paclitaxel with LY294002 was antagonistic in vitro when dexamethasone was also administered; although dexamethasone did not alter the Akt activity.
Activation of NFκB is linked to Akt-dependent transcription of pro-survival genes. Thus, LY294002-mediated suppression of the PI3K/Akt survival pathway with secondary inhibition of NFκB transcriptional activity is associated with enhancement of paclitaxel cytotoxicity in lung, esophageal and ovarian cancer cells[64,104,105], which indicates that NFκB may be the crucial intermediary step connecting Akt to the intrinsic susceptibility of cancer cells to paclitaxel.
Additionally, the Akt inhibitor MK-2206 augmented the efficacy of paclitaxel and carboplatin combination in gastric cancer, breast cancer, and melanoma cells. However, addition of MK-2206 to paclitaxel alone had no additive inhibitory effect on growth of nasopharyngeal carcinoma cells in vitro. Furthermore, Hirai et al found that synergy of MK-2206 with docetaxel was dependent on the treatment sequence, in which a schedule of MK-2206 before docetaxel was not effective in terms of growth inhibition. Dual inhibition of PI3K and mTORC1/2 by BEZ235 may overcome docetaxel resistance in human castration resistant prostate cancer in vitro and in vivo. Thus, modulation of the PI3K/Akt signaling may increase the efficacy and potency of taxanes according to in vitro and in vivo data. However, the effect may have been masked by inclusion of platinum in several studies, indicating that in some studies, the effect might be via platinum.
Effect of antimetabolites on Akt signaling
Antimetabolites are a large group of anticancer drugs widely used in combination therapy of various leukemias and solid tumors. They interfere with DNA and RNA synthesis and therefore the growth of tumor. Anti-metabolites are categorized as pyrimidine analogs [e.g., 5-fluorouracil (5-FU), gemcitabine], purine analogs (e.g., azathioprine, mercaptopurine), and antifolates (e.g., methotrexate, pemetrexed). In the present review, we mainly focused on two commonly used antimetabolites gemcitabine and 5-FU, as well as the novel anti-folate pemetrexed.
Effect of gemcitabine on Akt signaling and effect of Akt inhibition: Gemcitabine is used in the treatment of various carcinomas, such as lung cancer, bladder cancer, breast cancer, and lymphomas. A substantial number of potential biomarkers for sensitivity or resistance to gemcitabine have been characterized, including ribonucleotide reductase, deoxycytidine kinase, cytidine deaminase and human equilibrative transporter-1[112,113]. Additional mechanisms of resistance may exist, possibly not involving metabolism and direct targets[74,75,112,114].
Gemcitabine resistance in breast cancer cells may also be mediated by activation of the PI3K/Akt signaling pathway through phosphorylated Akt, so that inhibitors of PI3K/Akt might reverse the resistance to gemcitabine. Moreover, involvement and overexpression of PI3K and phosphorylated Akt in pancreatic carcinoma tissues has been reported in gemcitabine-resistant cells in vitro[73,78,84]. Rad51 overexpression may also mediate gemcitabine resistance through Akt or ERK1/2 activation in non-small cell lung cancer (NSCLC) cells, which could be overcome by downregulation of Rad51 or inhibition of Akt and ERK1/2 proteins. Although Akt phosphorylation status is tailored as a predictive biomarker for gemcitabine resistance in NSCLC patients, gemcitabine may also reduce Akt phosphorylation without affecting the Akt overall expression. Wilson et al reported a weak correlation between phosphorylated S6K and phosphorylated Akt, suggesting the existence of Akt-independent regulation of mTOR-mediated resistance to apoptosis. Overall, inhibition of PI3K/Akt signaling may enhance the gemcitabine cytotoxic profile.
Wortmannin enhanced the efficacy of gemcitabine by a 5-fold increase of apoptosis in murine pancreatic xenografts. A synergistic effect of Wortmannin, LY29 4002, and BEZ235 with gemcitabine was also reported in ovarian cancer and pancreatic carcinoma[74-77,81-85] in vitro and in vivo (Table 3). Although gemcitabine induces cell cycle arrest at the G1 and early S phases, PI3K/Akt activation does not seem to influence gemcitabine-induced cell cycle arrest. Likewise, perifosine has shown additivity in combination with gemcitabine by inhibiting Akt1 and Akt3 axis, and interfering Akt upstream, EGFR, and MET phosphorylation. Perifosine also enhanced the gemcitabine-mediated antitumor effect on pancreatic cancer cells through blocking p70S6K1 (S6K1) activation, and thus disrupting S6K1-Gli1 association and subsequent Gli1 activation. Besides, Akt, mTOR, and MAPK may also activate Gli1. Likewise, the Akt inhibitor MK2206 enhanced the effect of gemcitabine on growth inhibition in vitro and in vivo. In the contrary, Arlt et al found that NFκB, rather than PI3K/Akt, activity conferred resistance to gemcitabine in a panel of five pancreatic carcinoma cell lines, which was strongly diminished by NFκB inhibitors, and not by LY294002. Overall, the PI3K/Akt inhibitors have been efficacious in improving gemcitabine cytotoxicity.
Effect of FU on Akt signaling and effect of Akt inhibition: 5-FU is an antimetabolite that acts by inhibition of thymidylate synthase (TS) and can be incorporated into RNA and DNA altering the cancer cell replication and proliferation. 5-FU-based regimens are often used in adjuvant chemotherapy regimens and treatment of various advanced malignancies, such as colon cancer, head and neck cancer, breast cancer, but depending on the disease and stage of the tumor, intrinsic resistance to 5-FU can be as high as 50%. Resistance to 5-FU has often been associated with an increased TS expression, both transient and permanent. Other factors, such as enzymes involved in pyrimidine metabolism, i.e., increased dihydropyrimidine dehydrogenase, decreased orotate phosphoribosyltransferase, or altered folate metabolism have been associated with 5-FU resistance[121,124,125]. Moreover, 5-FU has major effects on glycosylation pathways as well, which may indirectly have effects on signaling pathways. Hence, evidence is accumulating that 5-FU resistance is associated with altered signaling.
Smad4 deficiency may also contribute to 5-FU resistance through upregulation of vascular endothelial growth factor expression, which is associated with increased vascular density[127,128]. Zhang et al found that loss of Smad4 in colorectal cancer patients may induce resistance to 5-FU through activation of Akt pathway. Akt can interact with Smad molecules to regulate transforming growth factor beta (TGF-β) signaling that is involved in transmitting chemical signals from the cell surface to the nucleus[130-132]. In summary, suppression of PI3K/Akt signaling may potentiate 5-FU.
The combination of LY294002 with 5-FU was synergistic via downregulation of PI3K/Akt signaling in Smad4-deficient colorectal cancer cells. Likewise, sequential combination of 5-FU and LY294002 induced synergistic cytotoxicity and overcame intrinsic and acquired resistance of 5-FU via downregulation of Akt and mitochondria-dependent apoptosis in an Epstein-Barr virus positive gastric cancer cell line. Wortmannin also promoted 5-FU antitumor activity in oral squamous cell carcinoma and breast cancer cells. In colorectal cancer cell lines, preclinical studies indicate that perifosine and BEZ235 may enhance the cytotoxic effects of 5-FU, likely through the NFκB and thus PI3K/Akt pathway. As a result, PI3K/Akt pathway is a rational target for sensitizing the tumor cells to 5-FU.
Effect of pemetrexed on Akt signaling and effect of Akt inhibition: Pemetrexed (Alimta; formerly known as LY231514), a multitargeted antifolate, inhibits thymidylate synthase (TS), dihydrofolate reductase, and the de novo purine nucleotide synthesis. Pemetrexed is currently used as a single agent, but more often in combination with cisplatin for first line treatment of non-squamous NSCLC and malignant pleural mesothelioma[138-140]. Resistance to pemetrexed has been associated with TS upregulation in a colon cancer cell line[123,141], a transport deficiency, decreased activation by folylpolyglutamate synthetase and increased efflux. Members of the ATP-binding cassette (ABC) transporters including P-glycoprotein (Pgp/ABCB1), multidrug resistance proteins (MRPs/ABCC) as well as breast cancer resistance protein (BCRP/ABCG2) as ATP-dependent drug efflux transporters may play roles in pemetrexed resistance. The PI3K/Akt pathway regulates ABCG2-mediated drug efflux, which induces drug resistance[86,143-145].
Pemetrexed can also activate Akt signaling[79,143,146,147], although its molecular mechanisms is not completely understood. Chen et al have observed a pemetrexed-induced cell apoptosis and a parallel increase in sustained Akt phosphorylation and nuclear accumulation in the NSCLC A549 cell line, and postulated that the activated Akt may play a proapoptotic role, while Giovannetti et al observed that pemetrexed increased EGFR phosphorylation and slightly reduced Akt phosphorylation and enhanced apoptosis in six NSCLC cell lines[143,146] and malignant pleural mesothelioma (MPM) cells, particularly when combined with EGFR inhibitor erlotinib or carboplatin.
Adding a PI3K/Akt inhibitor may further increase pemetrexed antineoplastic effect. LY294002 and Wortmannin decreased the pemetrexed-stimulated Akt and GSK3β phosphorylated activation in the NSCLC A549 cell line (Table 3). Perifosine antagonized the effect of pemetrexed in MPM cells by interfering upstream of Akt, affecting EGFR and MET phosphorylation. Likewise, BEZ235 enhanced the antineoplastic effect of pemetrexed in malignant pleural mesothelioma by decreasing ABCG2-mediated drug efflux at the cell surface, which may be of therapeutic value in combination regimens. These data suggest that combining pemetrexed with a PI3K/Akt inhibitor may result in a better antineoplastic effect in various tumors.
Effects of irradiation and chemoradiation on Akt signaling: The combination of radiation with cytotoxic chemotherapy has become a standard treatment option for the majority of inoperable, locally advanced cancers, including brain, head and neck, lung, and gastrointestinal malignancies. However, resistance to irradiation compromises therapeutic efficacy leading to tumor recurrence or metastasis. Tumors that recur after a successful radiation are often associated with radioresistance. Resistance to radiotherapy is predominantly related to efficient repair of the DNA damage induced by X-ray. Both normal and neoplastic cells have several types of repair pathways, usually starting with the recognition and excision of the lesion, and then insertion of a new nucleotide. Regulation of several of these repair enzymes is mediated through methylation of the gene or activation of various protein kinases. Given the complex biology underlying the interactions between the targeted agent and chemoradiation, comprehensive preclinical investigations are critical to design the rational combination.
Different combinations of drugs and radiation have been studied to improve efficacy and lessen toxicity. Chemotherapeutic drugs that perturb nucleotide metabolism have the potential to produce substantial sensitization of tumor cells to radiation treatment. Redistribution of cells into S-phase of the cell cycle and depletion of deoxynucleotide pools are probable mechanisms for gemcitabine and 5-FU, which made them potent radiosensitizers[151,152].
Radiation can activate multiple signaling pathways in cells, such as EGFR and several downstream proteins, i.e., PI3K/Akt, MAPK JNK, p38, NFκB, etc., stimulating DNA repair and thus causing radioresistance and survival of tumor cells. Loss of PTEN, as well as KRAS mutations[155,156] and NF-κB activation[157,158] also are associated with radioresistance, making the DNA less susceptible to ionizing radiation. Additionally, the ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, RAS mutation, and autocrine or paracrine ligands such as TGF-α, TGF-β, HB-EGF, neuregulins, and interleukin 6.
Effect of Akt-inhibition on radiation sensitivity: Alkylphosphocholines may potentiate the effect of radiation if given before or together with radiotherapy. Targeting the PI3K pathway by LY294002 led to radiosensitization in glioblastoma and human bladder cancer cell xenografts in vivo. BEZ235 has also shown a modest antitumor response in vivo, while the combination of BEZ235 and ionizing radiation provided a longer survival and led to a smaller tumor volume when compared to radiation alone. Likewise, the PI3K inhibitor BKM120 inhibited the radiation-induced activation of Akt. This induced suppression of DNA-double-strand breaks repair and increased apoptosis, which resulted in increased sensitivity of liver cancer cells to irradiation. Perifosine showed some radiosensitization in squamous cell carcinoma[163-165], malignant glioma, lymphoma, and prostate cancer. In contrast, one study failed to show any favorable results with perifosine in terms of increasing its anticancer potency, despite a significant reduction in the level of phosphorylated Akt as well as Akt activity in vitro and in vivo. Overall, given the activation of PI3K/Akt pathway by radiation, addition of a PI3K/Akt inhibitor may potentiate the therapeutic index of the conventional chemoradiation therapy.