mTOR Inhibitors Synergize on Regression, Reversal of Gene Expression, and Autophagy in Hepatocellular Carcinoma

Department of Cancer and Cell Biology, University of Cincinnati, Cincinnati, OH 45215, USA.
Science translational medicine (Impact Factor: 15.84). 04/2012; 4(139):139ra84. DOI: 10.1126/scitranslmed.3003923
Source: PubMed


Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Because mammalian target of rapamycin (mTOR) signaling is up-regulated in 50% of HCCs, we compared the effects of the U.S. Food and Drug Administration-approved mTOR-allosteric inhibitor, RAD001, with a new-generation phosphatidylinositol 3-kinase/mTOR adenosine triphosphate-site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting the proliferation of cultured HCC cells. The synergistic effect closely paralleled eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) dephosphorylation, which is implicated in the suppression of tumor cell proliferation. In a mouse model approximating human HCC, the drugs in combination, but not singly, induced a marked regression in tumor burden. However, in the tumor, BEZ235 alone was as effective as the combination in inhibiting 4E-BP1 phosphorylation, which suggests that additional target(s) may also be involved. Microarray analyses revealed a large number of genes that reverted to normal liver tissue expression in mice treated with both drugs, but not either drug alone. These analyses also revealed the down-regulation of autophagy genes in tumors compared to normal liver. Moreover, in HCC patients, altered expression of autophagy genes was associated with poor prognosis. Consistent with these findings, the drug combination had a profound effect on UNC51-like kinase 1 (ULK1) dephosphorylation and autophagy in culture, independent of 4E-BP1, and in parallel induced tumor mitophagy, a tumor suppressor process in liver. These observations have led to an investigator-initiated phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with HCC and other advanced solid tumors.

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    • "Both patients had some therapeutic benefit ranging from a PR to minor shrinkage per RECIST (-30%, -15%; respectively), which was maintained for 3.8 months and 8.3 months. According to the COSMIC database, with the exception of PIK3CA, none of the mutations putatively leading to PI3K/AKT/mTOR activation are commonly found in HCC; however, preclinical models and early clinical data generally suggest that targeting the PI3K/AKT/mTOR pathway can be an effective strategy in subsets of patients with HCC.[52, 53] However, none of the studies investigated these therapies in HCC with PI3K/AKT/mTOR activation.[54, 55] Revisiting such studies with gating of HCC patient for alterations in the PI3K/AKT/mTOR pathway may yield better evidence of the efficacy of inhibitors of the PI3K/mTOR pathway in this difficult to treat disease.[56-58] "
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    ABSTRACT: Understanding genetic aberrations in cancer leads to discovery of new targets for cancer therapies. The genomic landscape of hepatocellular carcinoma (HCC) has not been fully described. Therefore, patients with refractory advanced/metastatic HCC referred for experimental therapies, who had adequate tumor tissue available, had targeted next generation sequencing (NGS) of their tumor samples using the Illumina HiSeq 2000 platform (Foundation One, Foundation Medicine, MA) and their treatment outcomes were analyzed. In total, NGS was obtained for 14 patients (median number of prior therapies, 1) with advanced/metastatic HCC. Of these 14 patients, 10 (71%) were men, 4 (29%) women, 6 (43%) had hepatitis B or C-related HCC. NGS revealed at least 1 molecular abnormality in 12 patients (range 0-8, median 2). Detected molecular aberrations led to putative activation of the PI3K/AKT/mTOR pathway (n=3 [mTOR, PIK3CA, NF1]), Wnt pathway (n=6 [CTNNA1, CTNNB1]), MAPK pathway (n=2 [MAP2K1, NRAS]), and aberrant DNA repair mechanisms, cell cycle control and apoptosis (n=18 [ATM, ATR, BAP1, CCND1, CDKN2A, CDK4, FGF3, FGF4, FGF19, MCL1, MDM2, RB1, TP53]). Of the 3 patients with molecular aberrations putatively activating the PI3K/AKT/mTOR pathway, 2 received therapies including a mTOR inhibitor and all demonstrated therapeutic benefit ranging from a partial response to minor shrinkage per RECIST (-30%, -15%; respectively). In conclusion, genomic alterations are common in advanced HCC. Refractory patients with alterations putatively activating the PI3K/AKT/mTOR pathway demonstrated early signals of clinical activity when treated with therapies targeting mTOR.
    Oncotarget 05/2014; 5(10):3012-22. DOI:10.18632/oncotarget.1687 · 6.36 Impact Factor
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    • "Vertical blockade of the PI3K/Akt/mTOR pathway has been extensively studied to overcome the compensatory activation induced by single-agent treatment [24,28]. The synergistic anti-tumor effects were associated with a more sustained inhibition of PI3K/Akt/mTOR signaling activity and down-stream regulators of cell growth, including myc, cyclin D, and retinoblastoma (RB) protein. "
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    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 · 4.26 Impact Factor
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    • "It should be noted that this study unfortunately was performed in SK-HEP1 and its drug-resistant derivative (SK-Sora), which are derived from an HCC patient with ascites but are of endothelial origin. The combination of everolimus together with an AKT inhibitor (MK-2206), mTOR/PI3K inhibitor (BEZ235) or PI3K inhibitor (BKM120) showed improved anti-tumoral activity [77] [78] [80]. Further, improved efficacy was also achieved in vitro and in vivo with novel ATP-competitive mTOR kinase inhibitors together with histone deacetylase inhibitors [87]. "
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    ABSTRACT: Mechanistic target of rapamycin (mTOR) regulates cell growth, metabolism and aging in response to nutrients, cellular energy stage and growth factors. mTOR is frequently up-regulated in cancer including hepatocellular carcinoma (HCC) and is associated with bad prognosis, poorly differentiated tumors, and earlier recurrence. Blocking mTOR with rapamycin and first generation mTOR inhibitors, called rapalogs, has shown promising reduction of HCC tumors growth in preclinical models. Currently, rapamycin/rapalogs are used in several clinical trials for the treatment of advanced HCC, and as adjuvant therapy in HCC patients after liver transplantation and TACE. A second generation of mTOR pathway inhibitors has been developed recently, and is being tested in various clinical trials of solid cancers and has been used in preclinical HCC models. The results of series of clinical trials using mTOR inhibitors in HCC treatment will emerge in the near future.
    Journal of Hepatology 12/2013; 60(4). DOI:10.1016/j.jhep.2013.11.031 · 11.34 Impact Factor
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