Combined targeting of AKT and mTOR using MK-2206 and RAD001 is synergistic in the treatment of cholangiocarcinoma
ABSTRACT Cholangiocarcinoma (CCA) is a rare, but devastating disease arising from the epithelium of intra- and extrahepatic bile ducts. There are neither effective systemic therapies nor satisfying treatment options for inoperable CCA. Histopathological and biochemical studies of CCA show frequent dysregulation of the PI3K/AKT/mTOR pathway. Therefore, we investigated the efficacy of the mTOR inhibitor RAD001 and the impact of AKT signaling following mTOR inhibition in the treatment of CCA. RAD001 significantly inhibits proliferation of CCA cell lines, however, a concentration-dependent and isoform specific feedback activation of the three AKT isoforms (AKT1, AKT2, AKT3) was observed after mTOR inhibition. Since activation of AKT might limit the RAD001-mediated anti-tumor effect, the efficacy of combined mTOR and AKT inhibition was investigated using the allosteric AKT inhibitor MK-2206. Our results show that inhibition of AKT potentiates the efficacy of mTOR inhibition both in vitro and in a xenograft mouse model in vivo. Mechanistically, the anti-proliferative effect of the pan-AKT inhibitor MK2206 in the CCA cell line TFK-1 was due to inhibition of AKT1 and AKT2, because knockdown of either AKT1 or AKT2, but not AKT3, showed a synergistic reduction of cell proliferation in combination with mTOR treatment. Finally, by using an AKT isoform specific in vitro kinase assay, enzymatic activity of each of the three AKT isoforms was detected in all tissue samples from CCA patients, analyzed. In summary, our preclinical data suggest that combined targeting of mTOR and AKT using RAD001 and MK-2206 might be a new, effective strategy for the treatment of CCA. © 2013 Wiley Periodicals, Inc.
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- "For in vitro experiments, the drugs were dissolved in DMSO and the final DMSO concentration was kept below 0.1%. For in vivo experiments, the following the optimal dosage were used: NVP-AEW541 (dissolved in 25 mmol/L tartaric acid, 30 mg/kg, gavage) once per day for 25 days based on previous studies [22,23], BEZ235 (dissolved in 10% NMP (1-methyl-2-pyrrolidone)/PEG300 90%, 25 mg/kg, gavage) once per day for 25 days based on previous studies [24,25], and MK2206 (dissolved in 30% captisol, 100 mg/kg, i.p.) once per week for 4 weeks based on previous studies [26,27]. The antibodies used for Western blotting, and immunohistochemical staining included anti-Myc Tag (Origene, Rockville, MD), p-Akt, caspase 3, p-GSK3β, GSK3β, p-P70S6K, P70S6K, p-4EBP-1, Mcl-1, Bim, DR-5, FADD (Cell Signaling Technology, Danvers, MA), Bcl-2, Bad, Bax, survivin, GAPDH, and actin (Santa Cruz Biotechnology, Santa Cruz, CA). "
ABSTRACT: To explore whether combining inhibitors that target the insulin-like growth factor receptor (IGFR)/PI3K/Akt/mTOR signaling pathway (vertical blockade) can improve treatment efficacy for hepatocellular carcinoma (HCC). HCC cell lines (including Hep3B, Huh7, and PLC5) and HUVECs (human umbilical venous endothelial cells) were tested. The molecular targeting therapy agents tested included NVP-AEW541 (IGFR kinase inhibitor), MK2206 (Akt inhibitor), BEZ235 (PI3K/mTOR inhibitor), and RAD001 (mTOR inhibitor). Potential synergistic antitumor effects were tested by median dose-effect analysis in vitro and by xenograft HCC models. Apoptosis was analyzed by flow cytometry (sub-G1 fraction analysis) and Western blotting. The activities of pertinent signaling pathways and expression of apoptosis-related proteins were measured by Western blotting. Vertical blockade induced a more sustained inhibition of PI3K/Akt/mTOR signaling activities in all the HCC cells and HUVEC tested. Synergistic apoptosis-inducing effects, however, varied among different cell lines and drug combinations and were most prominent when NVP-AEW541 was combined with MK2206. Using an apoptosis array, we identified survivin as a potential downstream mediator. Over-expression of survivin in HCC cells abolished the anti-tumor synergy between NVP-AEW541 and MK2206, whereas knockdown of survivin improved the anti-tumor effects of all drug combinations tested. In vivo by xenograft studies confirmed the anti-tumor synergy between NVP-AEW541 and MK2206 and exhibited acceptable toxicity profiles. Vertical blockade of the IGFR/PI3K/Akt/mTOR pathway has promising anti-tumor activity for HCC. Survivin expression may serve as a biomarker to predict treatment efficacy.Molecular Cancer 01/2014; 13(1):2. DOI:10.1186/1476-4598-13-2 · 5.40 Impact Factor
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ABSTRACT: B-precursor acute lymphoblastic leukemia (B-pre ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. The prognosis of B-pre ALL has improved in pediatric patients, but the outcome is much less successful in adults. Constitutive activation of the PI3K/Akt/mTOR network is a feature of B-pre ALL, where it strongly influences cell growth and survival. RAD001, a selective mTORC1 inhibitor, has been shown to be cytotoxic against many types of cancer, including hematological malignancies. To investigate if mTORC1 could represent a target in the therapy of B-pre ALL, we treated cell lines and adult patient primary cells with RAD001. We documented that RAD001 decreased cell viability, induced cell cycle arrest in G0/G1 phase and caused apoptosis in B-pre ALL cell lines. Autophagy was also induced, that was important for the RAD001 cytotoxic effect, as down-regulation of Beclin-1 reduced drug cytotoxicity. RAD001 strongly synergized with the novel allosteric Akt inhibitor MK-2206, in both cell lines and patient samples. Similar results were obtained with the combination CCI-779 plus GSK 690693. These findings point out that mTORC1 inhibitors, either as a single-agent or in combination with Akt inhibitors, could represent a potential therapeutic innovative strategy in B-pre ALL.Leukemia accepted article preview online, 29 July 2013. doi:10.1038/leu.2013.226.Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 07/2013; 28(4). DOI:10.1038/leu.2013.226 · 9.38 Impact Factor
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ABSTRACT: Cholangiocarcinoma (CCA) is an orphan cancer with limited understanding of its genetic and genomic pathogenesis. Typically, it is highly treatment-refractory and patient outcome is dismal. Currently, there are no approved therapeutics for CCA and surgical resection remains the only option with curative intent. Clinical trials are currently being performed in a mixed cohort of biliary tract cancers that includes intrahepatic CCA, extrahepatic/perihilar CCA, distal extrahepatic CCA, gallbladder carcinoma and, in rare cases, even pancreatic cancers. Today, clinical trials fail primarily because they are underpowered mixed cohorts and designed without intent to enrich for markers to optimize success for targeted therapy. This review aims to emphasize current clinical attempts for targeted therapy of CCA, as well as highlight promising new candidate pathways revealed by translational genomics.01/2014; 1(1):143-157. DOI:10.2217/hep.13.4