sirolimus and dactolisib

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, acts as a "master switch" for cellular anabolic and catabolic processes, regulating the rate of cell growth and proliferation. Dysregulation of the mTOR signaling pathway occurs frequently in a variety of human tumors, and thus, mTOR has emerged as an important target for the design of anticancer agents. mTOR is found in two distinct multiprotein complexes within cells, mTORC1 and mTORC2. These two complexes consist of unique mTOR-interacting proteins and are regulated by different mechanisms. Enormous advances have been made in the development of drugs known as mTOR inhibitors. Rapamycin, the first defined inhibitor of mTOR, showed effectiveness as an anticancer agent in various preclinical models. Rapamycin analogues (rapalogs) with better pharmacologic properties have been developed. However, the clinical success of rapalogs has been limited to a few types of cancer. The discovery that mTORC2 directly phosphorylates Akt, an important survival kinase, adds new insight into the role of mTORC2 in cancer. This novel finding prompted efforts to develop the second generation of mTOR inhibitors that are able to target both mTORC1 and mTORC2. Here, we review the recent advances in the mTOR field and focus specifically on the current development of the second generation of mTOR inhibitors as anticancer agents.

Topics: Antineoplastic Agents; Cell Proliferation; Furans; Humans; Imidazoles; Indoles; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Morpholines; Multiprotein Complexes; Naphthyridines; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purines; Pyridines; Pyrimidines; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Aminopyridines; Animals; Apoptosis; Cell Line; Cytokines; Hippocampus; Imidazoles; Immunosuppressive Agents; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Morpholines; Neuroinflammatory Diseases; Oxidative Stress; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Seizures; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Glioblastomas (GBs) frequently display activation of the epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR). mTOR exists as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). In GBs, mTORC1 inhibitors such as rapamycin have performed poorly in clinical trials, and in vitro protect GB cells from nutrient and oxygen deprivation. Next generation ATP-competitive mTOR inhibitors with affinity for both mTOR complexes have been developed, but data exploring their effects on GB metabolism are scarce. In this study, we compared the ATP-competitive mTORC1/2 inhibitors torin2, INK-128 and NVP-Bez235 to the allosteric mTORC1 inhibitor rapamycin under conditions that mimic the glioma microenvironment. In addition to inhibiting mTORC2 signaling, INK-128 and NVP-Bez235 more effectively blocked mTORC1 signaling and prompted a stronger cell growth inhibition, partly by inducing cell cycle arrest. However, under hypoxic and nutrient-poor conditions mTORC1/2 inhibitors displayed even stronger cytoprotective effects than rapamycin by reducing oxygen and glucose consumption. Thus, therapies that arrest proliferation and inhibit anabolic metabolism must be expected to improve energy homeostasis of tumor cells. These results mandate caution when treating physiologically or therapeutically induced hypoxic GBs with mTOR inhibitors.

Topics: Apoptosis; Benzoxazoles; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Glioma; Humans; Imidazoles; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Naphthyridines; Protein Kinase Inhibitors; Pyrimidines; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral disc-largest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease.. mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitors-rapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitor-INK-128, PI3K&mTOR inhibitor-NVP-BEZ235, and Akt inhibitor-MK-2206-were applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated.. mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1β)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) -0.431 to -0.194; temsirolimus, 95% CI -0.529 to -0.292; everolimus, 95% CI -0.477 to -0.241; curcumin, 95% CI -0.248 to -0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-β-gal) positivity (versus rapamycin, 95% CI -0.437 to -0.230; temsirolimus, 95% CI -0.534 to -0.327; everolimus, 95% CI -0.485 to -0.278; curcumin, 95% CI -0.210 to -0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades.. mTORC1 inhibitors-notably temsirolimus with an improved water solubility-but not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Autophagy; Benzoxazoles; beta-Galactosidase; Cellular Senescence; Curcumin; Everolimus; Extracellular Matrix; Female; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Inflammation; Male; Matrix Metalloproteinases; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Microtubule-Associated Proteins; Middle Aged; Nucleus Pulposus; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Quinolines; Ribosomal Protein S6 Kinases, 70-kDa; Sequestosome-1 Protein; Sirolimus

To compare the anti-peritoneal fibrotic effects between a mammalian target of rapamycin complex 1-specific blocker and a phosphatidyl-inositol 3-kinase/mammalian target of rapamycin dual-blocker.. A total of 40 male Sprague-Dawley rats were randomly divided into five groups with eight animals per group. The normal group (N group) did not receive any intervention. The normal saline group (NS group) received an intraperitoneal injection of normal saline at 1 ml/100 g daily. The model group (3 W group), rapamycin (RAPA) group and BEZ235 (PI3K/mTOR dual-blocker) group all received an intraperitoneal injection of 0.1% chlorhexidine gluconate at 1 ml/100g daily. And the RAPA and BEZ235 groups also received a 0.5 mg/d RAPA or 2.5 mg/d BEZ235 gavage every day, respectively. Rats in each group were sacrificed after 3 weeks.. Immunohistochemistry, real-time PCR and western blotting analysis of fibrosis-related indicators (FN, Col 1, and α-SMA) confirmed that RAPA and BEZ235 significantly inhibited peritoneal fibrosis and that these two drugs had similar effects. The p-Akt, p-mTOR, p-p70S6K expression levels were significantly up-regulated in the 3 W group compared to the NS group, confirming that the mTOR pathway was significantly activated during peritoneal fibrosis. RAPA significantly inhibited the phosphorylation of mTOR and p70S6K but did not have significant effects on p-Akt upstream of mTOR. BEZ235 had significant inhibitory effects on all signaling molecules (p-Akt, p-mTOR, and p-p70S6K) in the mTOR pathway.. RAPA did not up-regulate p-Akt in a negative feedback fashion. Both drugs effectively inhibited peritoneal fibrosis.

Topics: Animals; Chlorhexidine; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; Imidazoles; Injections, Intraperitoneal; Kidney Failure, Chronic; Male; Mechanistic Target of Rapamycin Complex 1; Peritoneal Dialysis; Peritoneal Fibrosis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Quinolines; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Antineoplastic Agents; Chromones; Enzyme Inhibitors; Imidazoles; Kinetics; Mechanistic Target of Rapamycin Complex 1; Models, Biological; Morpholines; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; PTEN Phosphohydrolase; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

In the formation of goose fatty liver induced by a high-carbohydrate diet, it is characterized by the quick cell growth of liver. The carbohydrate is mostly digested and absorbed in the small intestine by the form of glucose. Recent studies have suggested a crucial role for PI3K-Akt-mTOR pathway in regulating cell proliferation, and then we speculate that PI3K-Akt-mTOR pathway may mediate glucose-induced liver cell proliferation. Goose primary hepatocytes were isolated and incubated in either no addition as a control or glucose or PI3K-Akt-mTOR pathway inhibitors or cotreatment with glucose and PI3K-Akt-mTOR pathway inhibitors. The results firstly showed that 35 mmol/l glucose stimulated the mRNA level and protein content of factors involved in PI3K-Akt-mTOR signal pathway in goose primary hepatocytes. Secondly, 35 mmol/l glucose evidently changed the cell cycle PI index and protein expression of cyclin D1. Meanwhile, the upregulation of 35 mmol/l glucose on the DNA synthesis rate, cell cycle PI index, the mRNA expression, protein content and protein expression of factors involved in the cell proliferation was decreased significantly by the inhibitors of PI3K-Akt-mTOR pathway, LY294002, rapamycin or NVP-BEZ235. In summary, glucose could stimulate the cell proliferation, and the PI3K-Akt-mTOR pathway inhibitors could dismiss glucose-induced the upregulation of cell proliferation in goose primary hepatocyte.

Topics: Animals; Anseriformes; Anti-Bacterial Agents; Antineoplastic Agents; Cell Proliferation; Cells, Cultured; Chromones; Enzyme Inhibitors; Gene Expression Regulation; Glucose; Hepatocytes; Imidazoles; Morpholines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Cancer stem cells (CSCs) have tumor initiation, self-renewal, metastasis and chemo-resistance properties in various tumors including colorectal cancer. Targeting of CSCs may be essential to prevent relapse of tumors after chemotherapy. Phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signals are central regulators of cell growth, proliferation, differentiation, and apoptosis. These pathways are related to colorectal tumorigenesis. This study focused on PI3K and mTOR pathways by inhibition which initiate differentiation of SW620 derived CSCs and investigated its effect on tumor progression. By using rapamycin, LY294002, and NVP-BEZ235, respectively, PI3K and mTOR signals were blocked independently or dually in colorectal CSCs. Colorectal CSCs gained their differentiation property and lost their stemness properties most significantly in dual-blocked CSCs. After treated with anti-cancer drug (paclitaxel) on the differentiated CSCs cell viability, self-renewal ability and differentiation status were analyzed. As a result dual-blocking group has most enhanced sensitivity for anti-cancer drug. Xenograft tumorigenesis assay by using immunodeficiency mice also shows that dual-inhibited group more effectively increased drug sensitivity and suppressed tumor growth compared to single-inhibited groups. Therefore it could have potent anti-cancer effects that dual-blocking of PI3K and mTOR induces differentiation and improves chemotherapeutic effects on SW620 human colorectal CSCs.

Topics: AC133 Antigen; Animals; Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Survival; Chromones; Colorectal Neoplasms; Humans; Imidazoles; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Neoplastic Stem Cells; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; SOXB1 Transcription Factors; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The PI3K/mTOR pathway is commonly deregulated in cancer. mTOR inhibitors are registered for the treatment of several solid tumors and novel inhibitors are explored clinically. Notably, this pathway also plays an important role in immunoregulation. While mTOR inhibitors block cell cycle progression of conventional T cells (Tconv), they also result in the expansion of CD4(+)CD25(hi)FOXP3(+) regulatory T cells (Tregs), and this likely limits their clinical antitumor efficacy. Here, we compared the effects of dual mTOR/PI3K inhibition (using BEZ235) to single PI3K (using BKM120) or mTOR inhibition (using rapamycin and everolimus) on Treg expansion and functionality. Whereas rapamycin, everolimus and BEZ235 effected a relative expansion benefit for Tregs and increased their overall suppressive activity, BKM120 allowed for similar expansion rates of Tregs and Tconv without altering their overall suppressive activity. Therefore, PI3K inhibition alone might offer antitumor efficacy without the detrimental selective expansion of Tregs associated with mTOR inhibition.

Topics: Aminopyridines; Antineoplastic Agents; Blotting, Western; Cell Proliferation; Cells, Cultured; Cytokines; Everolimus; Flow Cytometry; Humans; Imidazoles; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases

Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7,000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer has a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH‑2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. Using a wound healing assay we show that inhibition of the mTOR pathway using rapamycin, rapalogues, resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 h in these cell lines. We extended these findings up to 72 h with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Carcinoma; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Ovarian Neoplasms; Protein Kinase Inhibitors; Quinolines; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

In acute myeloid leukemia (AML), several signaling pathways such as the phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway are deregulated and constitutively activated as a consequence of genetic and cytogenetic abnormalities. We tested the effectiveness of PI3K/AKT/mTOR-targeting therapies and tried to identify alterations that associate with treatment sensitivity. By analyzing primary samples and cell lines, we observed a wide range of cytotoxic activity for inhibition of AKT (MK-2206), mTORC1 (rapamycin) and PI3K/mTORC1/2 (BEZ-235) with a high sensitivity of cells carrying an MLL rearrangement. In vivo PI3K/mTOR inhibition delayed tumor progression, reduced tumor load and prolonged survival in an MLL-AF9(+)/FLT3-ITD(+) xenograft mouse model. By performing targeted amplicon sequencing in 38 MLL-AF9(+) and 125 cytogenetically normal AML patient samples, we found a high additional mutation rate for genes involved in growth factor signaling in 79% of all MLL-AF9(+) samples, which could lead to a possible benefit of this cohort. PI3K/mTOR inhibition for 24 h led to the cross-activation of the ERK pathway. Further in vitro studies combining PI3K/mTOR and ERK pathway inhibition revealed highly synergistic effects in apoptosis assays. Our data implicate a possible therapeutic benefit of PI3K/mTOR inhibition in the MLL-mutated subgroup. Inhibiting rescue pathways could improve the therapeutic efficacy of PI3K-targeted therapies in AML.

Topics: Animals; Antineoplastic Agents; Apoptosis; Drug Synergism; Gene Expression Regulation, Leukemic; Gene Rearrangement; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mice; Myeloid-Lymphoid Leukemia Protein; Oncogene Proteins, Fusion; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; Survival Analysis; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

The clinical impact of HER2 inhibitors in the treatment of HER2-amplified breast cancers has been largely confined to chemotherapy combination regimens, since HER2 inhibitors appear to have very modest efficacies by themselves. This is due to the resilient nature of the functionally relevant HER2-HER3 tumor driver, bidirectionally linked with downstream PI3K/Akt pathway signaling, which can break through the inhibitory effects of most current HER2 or HER3 targeting therapies. A vertical combination approach targeting HER2 and a downstream pathway is a highly rational strategy for much more effective targeted therapy of this disease. However the importance of these downstream pathways in many human tissues and cells significant limits their usefulness as secondary targets by narrowing the therapeutic index of such combination therapies. The secondary target that can afford the highest potential for clinical translation is the one with the highest synergy against tumor cells in combination with HER2-inhibition, allowing the widest therapeutic index for clinical translation. We conducted a comparative analysis of such secondary targets in combination with the HER2 inhibitor lapatinib and find that the inhibition of mTor affords the highest degree of synergy. In further dissecting the individual roles of TORC1 and TORC2 complexes using pharmacologic and genetic tools, we find that it is specifically the inactivation of TORC2 that most synergistically enhances the efficacy of lapatinib. Although inhibitors that selectively target TORC2 are not currently available, these data make a compelling case for their development.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Drug Synergism; Humans; Imidazoles; Indoles; Lapatinib; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Naphthyridines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purines; Quinazolines; Quinolines; Receptor, ErbB-2; Receptor, ErbB-3; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

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.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Drug Synergism; Enzyme Inhibitors; Everolimus; Flow Cytometry; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Male; Mice, Inbred BALB C; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Quinolines; Receptors, Somatomedin; Signal Transduction; Sirolimus; Survivin; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis.. Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations.. We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation.. These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.

Topics: Antineoplastic Agents; Butadienes; Cell Cycle; Cell Line, Tumor; Chromones; Drug Screening Assays, Antitumor; Enzyme Activation; Everolimus; Humans; Imidazoles; Indazoles; MAP Kinase Signaling System; Mesothelioma; Molecular Targeted Therapy; Morpholines; Neoplasm Proteins; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; raf Kinases; Receptor Protein-Tyrosine Kinases; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Patients with clear cell carcinoma of the ovary (OCCC) have poor survival due to resistance to standard chemotherapy. OCCC has frequent activating mutations of the PIK3CA gene. The present study was conducted to clarify the efficacy of the inhibition of the PI3K-AKT-mTOR pathway in OCCC. We used 8 OCCC cell lines and 5 ovarian serous adenocarcinoma (OSAC) cell lines. The mutation status of the PIK3CA and KRAS genes was examined by direct sequencing. The IC50 values of NVP-BEZ235 (BEZ235) and temsirolimus were determined by WST-8 assay. Protein expression levels of PI3K-AKT-mTOR pathway molecules were examined by western blotting. Cell cycle distribution was analyzed by flow cytometry. Annexin V staining was used for detecting apoptosis. We also investigated the effects of BEZ235 on OCCC tumor growth in a nude mouse xenograft model. Four of the 8 OCCC cell lines showed a PIK3CA mutation while none of the 5 OSAC cell lines showed a mutation. The IC50 values of BEZ235 for the OCCC cell lines were lower than these values for the OSAC cell lines. The IC50 value of temsirolimus was higher than BEZ235 in the OCCC cell lines. The PIK3CA mutation was more frequently noted in OCCC than OSAC cells, but the sensitivity of these cell lines to BEZ235 or temsirolimus was not related to the mutation status. pHER3 and pAkt proteins were expressed more frequently in OCCC compared with OSAC. However, protein expression levels were distributed widely, and were not related to the sensitivity. Treatment with BEZ235 suppressed expression of pAkt, although treatment with temsirolimus did not. OCCC cells exhibited G1 phase arrest after treatment with BEZ235 and apoptosis with a higher concentration of the agent. BEZ235 significantly inhibited tumor growth in mice bearing OVISE and TU-OC-1 cell tumors. The present study indicated that the PI3K-AKT-mTOR pathway is a potential target for OCCC, and that BEZ235 warrants investigation as a therapeutic agent.

Topics: Adenocarcinoma, Clear Cell; Animals; Cell Line, Tumor; Cystadenoma, Serous; Female; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Mice; Mice, Nude; Neoplasms, Experimental; Ovarian Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Pancreatic endocrine tumors (PETs) are characterised by an indolent behaviour in terms of tumor growth. However, most patients display metastasis at diagnosis and no cure is currently available. Since the PI3K/AKT/mTOR axis is deregulated in PETs, the mTOR inhibitor RAD001 represents the first line treatment. Nevertheless, some patients do not respond to treatments and most acquire resistance. Inhibition of mTOR leads to feedback re-activation of PI3K activity, which may promote resistance to RAD001. Thus, PI3K represents a novel potential target for PETs. We tested the impact of three novel PI3K inhibitors (BEZ235, BKM120 and BYL719) on proliferation of PET cells that are responsive (BON-1) or unresponsive (QGP-1) to RAD001. BEZ235 was the most efficient in inhibiting proliferation in PET cells. Furthermore, combined treatment with BEZ235 and RAD001 exhibited synergic effects and was also effective in BON-1 that acquired resistance to RAD001 (BON-1 RR). Analysis of PI3K/AKT/mTOR pathway showed that RAD001 and BEZ235 only partially inhibited mTOR-dependent phosphorylation of 4EBP1. By contrast, combined therapy with the two inhibitors strongly inhibited phosphorylation of 4EBP1, assembly of the translational initiation complex and protein synthesis. Thus, combined treatment with BEZ235 may represent suitable therapy to counteract primary and acquired resistance to RAD001 in PETs.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Everolimus; Humans; Imidazoles; Neuroendocrine Tumors; Pancreatic Neoplasms; Phosphorylation; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Cellular signaling pathways involving mTOR, PI3K and ERK have dominated recent studies of breast cancer biology, and inhibitors of these pathways have formed a focus of numerous clinical trials. We have chosen trametinib, a drug targeting MEK in the ERK pathway, to address two questions. Firstly, does inhibition of a signaling pathway, as measured by protein phosphorylation, predict the antiproliferative activity of trametinib? Secondly, do inhibitors of the mTOR and PI3K pathways synergize with trametinib in their effects on cell proliferation? A panel of 30 human breast cancer cell lines was chosen to include lines that could be classified according to whether they were ER and PR positive, HER2 over-expressing, and "triple negative". Everolimus (targeting mTOR), NVP-BEZ235 and GSK2126458 (both targeting PI3K/mTOR) were chosen for combination experiments. Inhibition of cell proliferation was measured by IC50 values and pathway utilization was measured by phosphorylation of signaling kinases. Overall, no correlation was found between trametinib IC50 values and inhibition of ERK signaling. Inhibition of ERK phosphorylation was observed at trametinib concentrations not affecting proliferation, and sensitivity of cell proliferation to trametinib was found in cell lines with low ERK phosphorylation. Evidence was found for synergy between trametinib and either everolimus, NVP-BEZ235 or GSK2126458, but this was cell line specific. The results have implications for the clinical application of PI3K/mTOR and MEK inhibitors.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Everolimus; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Imidazoles; Inhibitory Concentration 50; MAP Kinase Signaling System; MCF-7 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridazines; Pyridones; Pyrimidinones; Quinolines; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis and drug resistance. Antitumor activity has been observed with mTOR inhibitors. However, they have shown limited clinical efficacy in relation to drug activation of feedback loops. Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage. Here, we have performed a comparative analysis of the mTOR inhibitor everolimus, the pan-PI3K inhibitor NVP-BKM120 and the dual PI3K/mTOR inhibitor NVP-BEZ235 in primary MCL cells. We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases. Among the three drugs, NVP-BEZ235 induced the highest change in gene expression profile. Functional validation demonstrated that NVP-BEZ235 inhibited angiogenesis, migration and tumor invasiveness in MCL cells. NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL. Our findings support the use of the dual PI3K/mTOR inhibitor NVP-BEZ235 as a promising approach to interfere with the microenvironment-related processes in MCL.

Topics: Aminopyridines; Cell Line, Tumor; Cell Proliferation; Chemokine CXCL12; Everolimus; Human Umbilical Vein Endothelial Cells; Humans; Imidazoles; Interleukin-4; Interleukin-6; Lymphoma, Mantle-Cell; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptome; Tumor Microenvironment

The major regulators of human acute lymphoblastic leukemia (ALL) cell growth and survival mediate their effects through the phosphoinositide 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. We have shown that the mTOR inhibitor everolimus extended survival in a non-obese diabetic/severe combined immune-deficient (NOD/SCID) mouse xenograft model of human ALL. Since PI-3K has mTOR dependent and independent functions we examined the effect of the dual PI-3K/mTOR inhibitors BEZ235 and BGT226. These agents inhibited the proliferation of ALL cell lines with a three log greater potency than everolimus. However, the induction of cell death differed, with BGT226 being cytotoxic in the low micromolar range while a two log higher concentration of BEZ235 was required to produce the same effect. While all three agents extended the survival of NOD/SCID mice engrafted with human ALL, the responses of individual xenografts varied. Although differential phosphorylation of AKT on Ser(473) and Thr(308) in response to everolimus exposure was observed, this did not entirely explain the different in vivo responses to the drugs. Our data suggests that while dual PI-3K/mTOR inhibitors may improve therapeutic outcomes for a subset of ALL patients, patient selection will be important, with some patients likely to respond better to single mTOR inhibition.

Topics: Animals; Cell Growth Processes; Cell Line, Tumor; Everolimus; Female; Humans; Imidazoles; In Vitro Techniques; Mice; Mice, SCID; Oncogene Protein v-akt; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Precision Medicine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

To evaluate first-generation rapamycin analogs (everolimus, temsirolimus, and rapamycin) and second-generation drugs inhibiting mTOR kinase (AZD-8055), PI3K (BKM-120) or both (BEZ-235 and GDC-0980) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) cells characterized for acquired resistance to sorafenib or sunitinib.. Anti-proliferative (MTT assay) and cell signaling (Western blot) effects of rapamycin analogs (1-20 μM) and second-generation drugs (0.03-20.0 μM) were assessed in human HCC SK-HEP1, RCC 786-0, and sorafenib- (SK-Sora) or sunitinib-resistant (786-Suni) cells.. In SK-HEP1 cells displaying high PTEN and Bcl2 expression, rapamycin analogs had poor anti-proliferative effects. However, SK-Sora cells were more sensitive to rapamycin analogs (≥1 μM) than SK-HEP1 cells. In 786-0 cells, lacking PTEN and Bcl2 expression, ≥1 μM rapamycin analogs blocked mTORC1 signaling, transiently activated Akt, and inhibited cell proliferation. Protracted sunitinib exposure in 786-Suni cells yielded an increase in p27 expression and a decreased sensitivity to rapamycin analogs, although mTORC1 function could be inhibited with rapamycin analogs. Second-generation drugs induced more potent growth inhibition than rapamycin analogs at concentrations >0.03 μM in parental cells, SK-Sora, and 786-Suni cells. Growth inhibitory concentrations of these new drugs also blocked mTORC1 downstream targets.. Rapamycin analogs inhibited mTORC1 downstream targets and yielded anti-proliferative effects in HCC and RCC cells. Second-generation drugs also appeared to be potent inhibitors of mTORC1 signaling; however, they appeared to be far more potent in inhibiting cellular proliferation in parental HCC and RCC cells and in cells developing resistance to sorafenib or sunitinib.

Topics: Aminopyridines; Antineoplastic Agents; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Everolimus; Humans; Imidazoles; Indoles; Kidney Neoplasms; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiprotein Complexes; Niacinamide; Phenylurea Compounds; Phosphoinositide-3 Kinase Inhibitors; Pyrimidines; Pyrroles; Quinolines; Signal Transduction; Sirolimus; Sorafenib; Sunitinib; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide with only few therapeutic options for patients with advanced disease. There is growing evidence indicating that activation of the PI3K/Akt/mTOR pathway plays an important role in HCC and therefore represents a promising target for novel therapeutic approaches. The aim of this study was to evaluate and compare the antitumour activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in vitro and in vivo.. The antitumour effects of RAD001, BEZ235 and BKM120 were analysed in seven hepatoma cell lines as mono and combination therapy with Doxorubicin, Cisplatin, Irinotecan or 5-Flourouracil in vitro and in xenografts. Cell-cycle progression, apoptosis, and autophagy were analysed. Furthermore, effects on mitochondrial respiration and glycolysis were assessed.. Treatment with RAD001, BEZ235 and BKM120 markedly reduced tumour cell viability. Combination of PI3K inhibitors with chemotherapy was most effective. RAD001, BEZ235 and BKM120 reduced tumour growth mainly by inhibiting cell-cycle progression rather than by inducing apoptosis. Interestingly, the antitumour effects were strongly associated with a reduction of mitochondrial respiration. BKM120, which exhibited the strongest antiproliferative effect, most strongly impaired oxidative phosphorylation compared with the other drugs.. In this study, BKM120 showed the strongest antitumour activity. Our findings suggest impairment of mitochondrial function as a relevant mechanism of BKM120. Moreover, combination of PI3K and mTOR inhibitors with cytotoxic agents could be promising option for non-cirrhotic HCC patients.

Topics: Aminopyridines; Analysis of Variance; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Camptothecin; Carcinoma, Hepatocellular; Cell Cycle; Cell Line; Cell Respiration; Cisplatin; Doxorubicin; Everolimus; Humans; Imidazoles; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Irinotecan; Liver Neoplasms; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

Mantle cell lymphoma (MCL) is an aggressive form of Non-Hodgkin-lymphoma (NHL) with an ongoing need for novel treatments. Apart from the translocation t(11:14), which facilitates constitutive transcription of cyclin D1, additional aberrations are frequently observed in MCL, including a recurrent dysregulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. mTOR, a key component of this pathway, is pivotal for the assembly of mTOR complex (mTORC) 1 and 2. Temsirolimus, an analog of the mTOR inhibitor rapamycin, is approved for the treatment of relapsed MCL. Response rates, however, are low and response durations are short. We demonstrate that inhibition of mTORC1 by rapamycin or blocking of mTORC1 and mTORC2 in conjunction with PI3K by NVP-BEZ235 reduces proliferation of MCL cell lines to a similar extent. However, only NVP-BEZ235 is able to sufficiently inhibit the downstream pathway of mTOR and to mediate cell death through activation of the intrinsic apoptosis pathway. Further analysis demonstrated that the anti-apoptotic Bcl-2 family member Mcl-1 plays a central role in regulation of MCL survival. While Mcl-1 protein levels remained unchanged after coculture with rapamycin, they were down-regulated in NVP-BEZ235 treated cells. Furthermore, inhibition of Mcl-1 by the BH3-only mimetic obatoclax or down-regulation of constitutive Mcl-1, but not of Bcl-2 or Bcl-xL, by siRNA facilitated cell death of MCL cells and enhanced NVP-BEZ235's capacity to induce cell death. Our findings may help to lay the foundation for further improvements in the treatment of MCL.

Topics: Amino Acid Chloromethyl Ketones; Antibiotics, Antineoplastic; Apoptosis; bcl-X Protein; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Humans; Imidazoles; Indoles; Lymphoma, Mantle-Cell; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Myeloid Cell Leukemia Sequence 1 Protein; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Quinolines; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Bronchial carcinoids (BCs) are rare neuroendocrine tumors that are still orphans of medical treatment. Human BC primary cultures may display resistance to everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), in terms of cell viability reduction. Our aim was to assess whether the novel dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor NVP-BEZ235 is effective in everolimus-resistant human BC tissues and cell lines. In addition, we searched for possible markers of the efficacy of mTOR inhibitors that may help in identifying the patients who may benefit from treatment with mTOR inhibitors, sparing them from ineffective therapy. We found that NVP-BEZ235 is twice as potent as everolimus in reducing cell viability and activating apoptosis in human BC tissues that display sensitivity to mTOR inhibitors, but is not effective in everolimus-resistant BC tissues and cell lines that bypass cyclin D1 downregulation and escape G0/G1 blockade. Rebound AKT activation was not observed in response to treatment with either mTOR inhibitor in the 'resistant' BC cells. In addition to total mTOR levels, putative markers of the sensitivity of BCs to mTOR inhibitors are represented by AKT, p70S6K (RPS6KB2), and ERK1/2 (MAPK3/1) protein levels. Finally, we validated these markers in an independent BC group. These data indicate that the dual PI3K/mTOR inhibitor NVP-BEZ235 is more potent than everolimus in reducing the proliferation of human BC cells. 'Resistant' cells display lower levels of mTOR, p70S6K, AKT, and ERK1/2, indicating that these proteins may be useful as predictive markers of resistance to mTOR and PI3K/mTOR inhibitors in human BCs.

Topics: Adult; Aged; Antineoplastic Agents; Carcinoid Tumor; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Imidazoles; Lung Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Young Adult

Everolimus, an mTOR inhibitor, showed great clinical efficacy in combination with tamoxifen, letrozole, or exemestane for the treatment of estrogen receptor-positive (ER+) breast cancer. However, its antitumor activity was shown to be compromised by a compensatory process involving AKT activation. Here, it was determined whether combining an additional PI3K inhibitor can reverse this phenomenon and improve treatment efficacy. In breast cancer cells (MCF-7 and BT474), everolimus inhibited the mTOR downstream activity by limiting phosphorylation of p70S6K and 4EBP1, which resulted in p-Ser473-AKT activation. However, addition of a LY294002, a PI3K inhibitor, to tamoxifen and everolimus treatment improved the antitumor effect compared with tamoxifen alone or the other two agents in combination. Moreover, LY294002 suppressed the activity of the PI3K/AKT/mTOR axis and mitigated the p-Ser473-AKT activation feedback loop in both cell lines. Critically, this combination scheme also significantly inhibited the expression of HIF-1a, an angiogenesis marker, under hypoxic conditions and reduced blood vessel sprout formation in vitro. Finally, it was shown that the three-agent cocktail had the greatest efficacy in inhibiting MCF-7 xenograft tumor growth and angiogenesis. Taken together, these results suggest that inhibition of PI3K and mTOR may further improve therapy in ER(+) breast cancer cells.. Combinatorial inhibition of the PI3K/AKT/mTOR signaling axis may enhance endocrine-based therapy in breast cancer.

Topics: Aminopyridines; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Everolimus; Female; Humans; Imidazoles; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; Tamoxifen; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Posttransplant lymphoproliferative disorder (PTLD) continues to be a devastating and potentially life-threatening complication in organ transplant recipients. PTLD is associated with EBV infection and can result in malignant B cell lymphomas. Here we demonstrate that the PI3K/Akt/mTOR pathway is highly activated in EBV+ B cell lymphoma lines derived from patients with PTLD. Treatment with the mTORC1 inhibitor Rapamycin (RAPA) partially inhibited the proliferation of EBV+ B cell lines. Resistance to RAPA treatment correlated with high levels of Akt phosphorylation. An mTORC1/2 inhibitor and a PI3K/mTOR dual inhibitor suppressed Akt phosphorylation and showed a greater anti-proliferative effect on EBV+ B lymphoma lines compared to RAPA. EBV+ B cell lymphoma lines expressed high levels of PI3Kδ. We demonstrate that PI3Kδ is responsible for Akt activation in EBV+ B cell lymphomas, and that selective inhibition of PI3Kδ by either siRNA, or a small molecule inhibitor, augmented the anti-proliferative effect of RAPA on EBV+ B cell lymphomas. These results suggest that PI3Kδ is a novel, potential therapeutic target for the treatment of EBV-associated PTLD and that combined blockade of PI3Kδ and mTOR provides increased efficacy in inhibiting proliferation of EBV+ B cell lymphomas.

Topics: Blotting, Western; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Imidazoles; Lymphoma, B-Cell; Lymphoproliferative Disorders; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Organ Transplantation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Postoperative Complications; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established.. We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative.. Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2.. Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.

Topics: Aminopyridines; Antineoplastic Agents; Drug Synergism; Everolimus; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lymphocytes; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Morpholines; Multiprotein Complexes; Naphthyridines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Pyrimidines; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is frequently dysregulated in diffuse large B cell lymphoma (DLBCL) including the favorable germinal centre B-cell (GCB) and the unfavorable activated B-cell (ABC) subtypes. mTOR promotes cap-dependent translation of proteins, like Mcl-1, through inhibitory phosphorylation of the eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Inhibition of mTOR by RAD001 reduces proliferation but fails to dephosphorylate 4EBP1 and to induce cell death in either DLBCL subtype. In contrast, concurrent inhibition of PI3K and mTOR with NVP-BEZ235 inhibits proliferation, dephosphorylates 4EBP1, and induces cells death, notably more pronounced in CGB cells. Small RNA interference identifies Mcl-1 as a crucial cell death mediator of both DLBCL subtypes. Inhibition of the PI3K/mTOR/4EBP1 by NVP-BEZ235 results in suppression of the cap-dependent translation initiation complex and concomitant downregulation of Mcl-1 in GCB cell lines. In ABC cell lines, this suppression is possibly compensated by NF-κB- or Pim kinase-mediated signaling.

Topics: Adaptor Proteins, Signal Transducing; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Death; Cell Line, Tumor; Everolimus; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lymphoma, Large B-Cell, Diffuse; Myeloid Cell Leukemia Sequence 1 Protein; NF-kappa B; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-pim-1; Quinolines; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a significant public health concern in regions where they are endemic. On the other hand, evidence indicates that the globally distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway participates in many cellular processes, including cell survival and differentiation, and also has been shown to play important roles in different steps of the life cycles of a variety of viruses. Here we report that the inhibition of the PI3K/Akt pathway inhibited budding and to a lesser extent RNA synthesis, but not cell entry, of LCMV. Accordingly, BEZ-235, a PI3K inhibitor currently in cancer clinical trials, inhibited LCMV multiplication in cultured cells. These findings, together with those previously reported for Junin virus (JUNV), indicate that targeting the PI3K/Akt pathway could represent a novel antiviral strategy to combat human-pathogenic arenaviruses.

Topics: Animals; Arenavirus; Cell Line; Chlorocebus aethiops; Chromones; Gene Expression Regulation, Viral; Humans; Imidazoles; Lassa virus; Lymphocytic choriomeningitis virus; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; RNA, Viral; Signal Transduction; Sirolimus; Virus Internalization; Virus Release; Virus Replication

The mammalian target of rapamycin (mTOR) is frequently activated in colon cancers due to mutations in the phosphatidylinositol 3-kinase (PI3K) pathway. Targeting mTOR with allosteric inhibitors of mTOR such as rapamycin reduces colon cancer progression in several experimental models. Recently, a new class of mTOR inhibitors that act as ATP-competitive inhibitors of mTOR, has been developed. The effectiveness of these drugs in colon cancer cells has however not been fully characterized.. LS174T, SW480 and DLD-1 colon cancer cell lines were treated with PP242 an ATP-competitive inhibitor of mTOR, NVP-BEZ235, a dual PI3K/mTOR inhibitor or rapamycin. Tumor cell growth, proliferation and survival were assessed by MTS assay, 5-bromo-2'-deoxyuridine (BrDU) incorporation or by quantification of DNA fragmentation respectively. In vivo, the anticancer activity of mTOR inhibitors was evaluated on nude mice bearing colon cancer xenografts.. PP242 and NVP-BEZ235 reduced the growth, proliferation and survival of LS174T and DLD-1 colon cancer cells more efficiently than rapamycin. Similarly, PP242 and NVP-BEZ235 also decreased significantly the proliferation and survival of SW480 cells which were resistant to the effects of rapamycin. In vivo, PP242 and NVP-BEZ235 reduced the growth of xenografts generated from LS174T and SW480 cells. Finally, we also observed that the efficacy of ATP-competitive inhibitors of mTOR was enhanced by U0126, a MEK inhibitor.. Taken together, these results show that ATP-competitive inhibitors of mTOR are effective in blocking colon cancer cell growth in vitro and in vivo and thus represent a therapeutic option in colon cancer either alone or in combination with MEK inhibitors.

Topics: Animals; Antibiotics, Antineoplastic; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Female; Humans; Imidazoles; Immunohistochemistry; Indoles; Male; Mice; Protein Kinase Inhibitors; Purines; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Development of resistance to endocrine therapy is a clinical issue in estrogen receptor (ER)-positive breast cancer. Here we show that persistent activation of AKT/mTOR signaling is crucial to the acquisition of letrozole resistance in cell clones generated from MCF-7/AROM-1 aromatase-expressing breast cancer cells after prolonged letrozole exposure. ERα plays a marginal role in this context. As a proof of concept, the association between PI3K/AKT/mTOR signaling and insensitivity to endocrine therapies was confirmed in breast cancer patients who developed early letrozole resistance in neoadjuvant setting. In addition our results suggest that, regardless of the mechanism mediating the activation of AKT/mTOR pathway, either RAD001 or NVP-BEZ235 treatment may represent a promising strategy to overcome acquired resistance to letrozole in breast cancers dependent on AKT/mTOR signaling.

Topics: Aged; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Imidazoles; Immunosuppressive Agents; Letrozole; Neoadjuvant Therapy; Nitriles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triazoles

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.

Topics: Animals; Antineoplastic Agents; Autophagy; Autophagy-Related Protein-1 Homolog; Carcinoma, Hepatocellular; Everolimus; Humans; Imidazoles; Immunoblotting; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Protein Serine-Threonine Kinases; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235--a dual PI3K/mTOR inhibitor--and RAD001--an mTOR inhibitor--in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n = 9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n = 4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Agents; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Dose-Response Relationship, Drug; Endometrial Neoplasms; Everolimus; Female; G1 Phase Cell Cycle Checkpoints; Genotype; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Imidazoles; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Quinolines; ras Proteins; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Currently, there is no completely effective therapy available for metastatic phaeochromocytomas (PCCs) and paragangliomas. In this study, we explore new molecular targeted therapies for these tumours, using one more benign (mouse phaeochromocytoma cell (MPC)) and one more malignant (mouse tumour tissue (MTT)) mouse PCC cell line - both generated from heterozygous neurofibromin 1 knockout mice. Several PCC-promoting gene mutations have been associated with aberrant activation of PI3K/AKT, mTORC1 and RAS/RAF/ERK signalling. We therefore investigated different agents that interfere specifically with these pathways, including antagonism of the IGF1 receptor by NVP-AEW541. We found that NVP-AEW541 significantly reduced MPC and MTT cell viability at relatively high doses but led to a compensatory up-regulation of ERK and mTORC1 signalling at suboptimal doses while PI3K/AKT inhibition remained stable. We subsequently investigated the effect of the dual PI3K/mTORC1/2 inhibitor NVP-BEZ235, which led to a significant decrease of MPC and MTT cell viability at doses below 50 nM but again increased ERK signalling. Accordingly, we next examined the combination of NVP-BEZ235 with the established agent lovastatin, as this has been described to inhibit ERK signalling. Lovastatin alone significantly reduced MPC and MTT cell viability at therapeutically relevant doses and inhibited both ERK and AKT signalling, but increased mTORC1/p70S6K signalling. Combination treatment with NVP-BEZ235 and lovastatin showed a significant additive effect in MPC and MTT cells and resulted in inhibition of both AKT and mTORC1/p70S6K signalling without ERK up-regulation. Simultaneous inhibition of PI3K/AKT, mTORC1/2 and ERK signalling suggests a novel therapeutic approach for malignant PCCs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Survival; Dose-Response Relationship, Drug; Everolimus; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Lovastatin; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; PC12 Cells; Protein Kinase Inhibitors; Proteins; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Quinolines; Receptor, IGF Type 1; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Interleukin 22 (IL-22), a relatively new cytokine has been found to induce significant proliferation of human keratinocytes and fibroblast like synoviocytes (FLS) and thus plays an important role in the pathogenesis of autoimmune diseases like psoriasis and rheumatoid arthritis (RA) which are characterized by hyperproliferation of keratinocytes and FLS respectively. PI3K/Akt/mTOR signaling cascade plays crucial role in cell growth and survival. Therefore our objective was to see the regulatory role of PI3K/Akt/mTOR signaling cascade in IL-22 induced proliferation of keratinocytes and FLS.. Normal human epidermal keratinocytes (NHEK) and FLS were isolated from skin of healthy volunteer's undergone plastic surgery and synovial tissue of psoriatic arthritis (PsA) and RA patients respectively. IL-22 induced proliferation of NHEK and FLS was measured by MTT assay. Phosphorylation of Akt/mTOR was determined by western blot assay and further confirmed by real time polymerase chain reaction (RT-PCR).. We observed that IL-22 induced significant proliferation of NHEK and FLS which was effectively inhibited by dual kinase (PI3K/mTOR) inhibitor, NVP-BEZ235 and specific mTOR inhibitor, Rapamycin. In NHEK and FLS, IL-22 significantly induced phosphorylation of Akt and mTOR which was effectively blocked by Rapamycin and NVP-BEZ235. Further we did RT-PCR in NHEK and found that IL-22 significantly upregulated AKT1 and MTOR gene.. These results show that IL-22 induced proliferation of NHEK and FLS is dependent on PI3K/Akt/mTOR signaling pathway. This novel observation provides the scope to develop new therapeutics targeting PI3K/Akt/mTOR signaling pathway in autoimmune diseases like psoriasis and rheumatoid arthritis.

Topics: Arthritis; Arthritis, Rheumatoid; Blotting, Western; Cell Proliferation; Cells, Cultured; Fibroblasts; Humans; Imidazoles; Interleukin-22; Interleukins; Keratinocytes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Psoriasis; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; Synovial Membrane; TOR Serine-Threonine Kinases

Mantle cell lymphoma (MCL) is a distinct subtype of B-cell lymphoma; the complete response rate for standard therapies in use today is 85 - 90%. NVP-BEZ235 inhibits the PI3K/Akt/mTOR signaling axis at the level of both PI3K and mTOR. In this study, we analyzed the inhibitory effects of NVP-BEZ235 on mantle cell lines and its effects in combination with enzastaurin, everolimus and perifosine.. The effects of NVP-BEZ235 on cell proliferation and apoptosis were evaluated using MTT assay and flow cytometry analysis. The cell cycle analysis was performed applying BrdU incorporation. Western blot analysis was utilized for phosphorylation status evaluation of protein kinases. The interaction between NVP-BEZ235 and enzastaurin, everolimus and perifosine was examined by Chou-Talalay method.. NVP-BEZ235 induced significant increase of apoptosis, both via intrinsic and extrinsic pathways. We found that NVP-BEZ235 inhibited mantle cells growth by induction of G1 arrest. NVP-BEZ235 exerts its antitumor activity even when mantle cells were in contact with bone marrow microenvironment. Enzastaurin, everolimus and perifosine enhanced the cytotoxicity triggered by NVP-BEZ235.. The above results encourage clinical development of NVP-BEZ235 in combination and the possible inclusion of patients with mantle lymphoma in Phase I/II clinical trials.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Bone Marrow; Cell Line, Tumor; Cell Proliferation; Everolimus; Flow Cytometry; G1 Phase Cell Cycle Checkpoints; Humans; Imidazoles; Indoles; Lymphoma, Mantle-Cell; Phosphorylation; Phosphorylcholine; Protein Kinases; Quinolines; Sirolimus

Adrenocortical cancer is a rare malignancy for which current pharmacological therapies are still insufficient. We tested the effect of a novel PI3 kinase - mammalian target of rapamycin dual inhibitor (NVP-BEZ235) on proliferation of the H295R adrenocortical cancer cell line in vitro and grown as xenografts in immunodeficient mice. NVP-BEZ235 was able to significantly inhibit phosporylation of Akt kinase and S6 ribosomal protein in H295R cells and to significantly reduce their proliferation in vitro and xenograft growth in vivo. The drug also induced activation of Erk phosphorylation, which could be inhibited by simultaneous treatment with the Erk inhibitor FR180204. This latter drug synergized with NVP-BEZ235 in the inhibition of H295R proliferation in vitro. Our data suggest that dual PI3K/mTOR inhibitors may represent a useful pharmacological tool in the therapy of advanced adrenocortical cancer and that simultaneous inhibition of both Erk and PI3K - mTOR pathways may be required to obtain a higher antiproliferative effect in this type of tumor.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Drug Evaluation, Preclinical; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Mice; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Pyridazines; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Xenograft Model Antitumor Assays

The mammalian target of rapamycin (mTOR) is regulated by oncogenic growth factor signals and plays a pivotal role in controlling cellular metabolism, growth and survival. Everolimus (RAD001) is an allosteric mTOR inhibitor that has shown marked efficacy in certain cancers but is unable to completely inhibit mTOR activity. ATP-competitive mTOR inhibitors such as NVP-BEZ235 can block rapamycin-insensitive mTOR readouts and have entered clinical development as anti-cancer agents. Here, we show the degree to which RAD001 and BEZ235 can be synergistically combined to inhibit mTOR pathway activation, cell proliferation and tumor growth, both in vitro and in vivo. RAD001 and BEZ235 synergized in cancer lines representing different lineages and genetic backgrounds. Strong synergy is seen in neuronal, renal, breast, lung, and haematopoietic cancer cells harboring abnormalities in PTEN, VHL, LKB1, Her2, or KRAS. Critically, in the presence of RAD001, the mTOR-4EBP1 pathway and tumorigenesis can be fully inhibited using lower doses of BEZ235. This is relevant since RAD001 is relatively well tolerated in patients while the toxicity profiles of ATP-competitive mTOR inhibitors are currently unknown.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Drug Synergism; Everolimus; Humans; Imidazoles; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The tumour-selective death receptor ligand tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for the treatment of human cancer. However, many tumours have evolved mechanisms to resist TRAIL-induced apoptosis. A number of studies have demonstrated that aberrant PI(3)K-Akt-mTOR survival signalling may confer TRAIL resistance by altering the balance between pro- and anti-apoptotic proteins. Here, we show that neuroendocrine tumour (NET) cell lines of heterogeneous origin exhibit a range of TRAIL sensitivities and that TRAIL sensitivity correlates with the expression of FLIP(S), caspase-8, and Bcl-2. Neither single mTOR inhibition by everolimus nor dual mTOR/PI(3)K inhibition by NVP-BEZ235 was able to enhance TRAIL susceptibility in any of the tested cell lines. In contrast, dual PI(3)K-Akt-mTOR and Raf-MEK-Erk pathway inhibition by the IGF-1R inhibitor NVP-AEW541 effectively restored TRAIL sensitivity in NCI-H727 bronchus carcinoid cells. Furthermore, blocking Raf-MEK-Erk signalling by the novel Raf inhibitor Raf265 significantly enhanced TRAIL sensitivity in NCI-H727 and CM insulinoma cells. While having no effect on FLIP(S) or caspase-8 expression, Raf265 strongly decreased Bcl-2 levels in those cell lines susceptible to its TRAIL-sensitizing action. Taken together, our findings suggest that combinations of Raf-MEK-Erk pathway inhibitors and TRAIL might offer a novel therapeutic strategy in NET disease.

Topics: Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Enzyme Inhibitors; Everolimus; Humans; Imidazoles; Neuroendocrine Tumors; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-bcl-2; Pyridines; Quinolines; Receptor, IGF Type 1; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; TNF-Related Apoptosis-Inducing Ligand; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.

Topics: Angiogenesis Inhibitors; Animals; Butadienes; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Humans; Imidazoles; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Morpholines; Multiprotein Complexes; Neoplasms; Neovascularization, Pathologic; Nitriles; Proteins; Pyrimidines; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

The phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit PI3K, mTOR or both are currently under development. The mTOR allosteric inhibitor, RAD001, and the PI3K/mTOR dual kinase inhibitor, BEZ235, are examples of these agents. We were interested in developing strategies to enhance mTOR-targeted caner therapy. In this study, we found that BEZ235 alone effectively inhibited the growth of rapamycin-resistant cancer cells. Interestingly, the combination of sub-optimal concentrations of RAD001 and BEZ235 exerted synergistic inhibition of the growth of human lung cancer cells along with induction of apoptosis and G1 arrest. Furthermore, the combination was also more effective than either agent alone in inhibiting the growth of lung cancer xenografts in mice. The combination showed enhanced effects on inhibiting mTOR signaling and reducing the expression of c-Myc and cyclin D1. Taken together, our results suggest that the combination of RAD001 and BEZ235 is a novel strategy for cancer therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Eukaryotic Initiation Factor-4F; Everolimus; Female; G1 Phase; Humans; Imidazoles; Lung Neoplasms; Mice; Proto-Oncogene Proteins c-myc; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

PI3K and mTOR are key components of signal transduction pathways critical for cell survival. Numerous PI3K inhibitors have entered clinical trials, while mTOR is the target of approved drugs for metastatic renal cell carcinoma (RCC). We characterized expression of p85 and p110α PI3K subunits and mTOR in RCC specimens and assessed pharmacologic co-targeting of these molecules in vitro.. We employed tissue microarrays containing 330 nephrectomy cases using a novel immunofluorescence-based method of Automated Quantitative Analysis (AQUA) of in situ protein expression. In RCC cell lines we assessed synergism between PI3K and mTOR inhibitors and activity of NVP-BEZ235, which co-targets PI3K and mTOR.. p85 expression was associated with high stage and grade (P < 0.0001 for both). High p85 and high mTOR expression were strongly associated with decreased survival, and high p85 was independently prognostic on multi-variable analysis. Strong co-expression of both PI3K subunits and mTOR was found in the human specimens. The PI3K inhibitor LY294002 and rapamycin were highly synergistic in all six RCC cell lines studied. Similar synergism was seen with all rapamycin concentrations used. NVP-BEZ235 inhibited RCC cell growth in vitro with IC(50)s in the low ηM range and resultant PARP cleavage.. High PI3K and mTOR expression in RCC defines populations with decreased survival, suggesting that they are good drug targets in RCC. These targets tend to be co-expressed, and co-targeting these molecules is synergistic. NVP-BEZ235 is active in RCC cells in vitro; suggesting that concurrent PI3K and mTOR targeting in RCC warrants further investigation.

Topics: Apoptosis; Automation; Biomarkers, Tumor; Carcinoma, Renal Cell; Cell Line, Tumor; Chromones; Drug Synergism; Humans; Imidazoles; Inhibitory Concentration 50; Kidney Neoplasms; Molecular Targeted Therapy; Morpholines; Multivariate Analysis; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; Sirolimus; TOR Serine-Threonine Kinases

Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.

Topics: Blotting, Western; Cell Cycle; Cell Division; Cell Line, Tumor; Drug Synergism; Humans; Imidazoles; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Triazines

Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Carcinoma, Transitional Cell; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Everolimus; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Phosphoproteins; Proto-Oncogene Proteins c-akt; Quinolines; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Several studies have established a link between aberrant PI(3)K-Akt-mTOR- and Ras-Raf-MEK-Erk1/2 signaling and neuroendocrine tumor disease. In this study, we comparatively investigate the antitumor potential of novel small-molecule inhibitors targeting mTOR (RAD001), mTOR/PI(3)K (NVP-BEZ235) and Raf (Raf265) on human NET cell lines of heterogeneous origin. All inhibitors induced potent antitumor effects which involved the induction of apoptosis and G0/G1 arrest. However, the dual mTOR/PI(3)K inhibitor NVP-BEZ235 was more efficient compared to the single mTOR inhibitor RAD001. Consistently, NVP-BEZ235 prevented the negative feedback activation of Akt as observed after treatment with RAD001. Raf265 inhibited Erk1/2 phosphorylation but strongly induced Akt phosphorylation and VEGF secretion, suggesting the existence of a compensatory feedback loop on PI3K-Akt signaling. Finally, combined treatment with RAD001 or NVP-BEZ235 and Raf265 was more efficient than single treatment with either kinase inhibitor. Together, our data provide a rationale for dual targeting of PI(3)K-Akt-mTOR- and Ras-Raf-MEK-Erk1/2 signaling in NET disease.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Everolimus; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; G1 Phase; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Neuroendocrine Tumors; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Quinolines; raf Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Inhibitors of TORC1 have been shown to be active in patients with metastatic renal cell carcinoma (RCC). As the phosphatidylinositol 3-kinase (PI3K) pathway activates numerous other kinases, transcription factors, and proteins associated with cell growth and survival besides mammalian target of rapamycin (mTOR), disruption of this pathway upstream of mTOR may be more effective than inhibition of TORC1 alone.. To investigate this possibility, the dual PI3K/mTOR inhibitor NVP-BEZ235 was compared with rapamycin in RCC cell lines and xenografts generated from 786-O and A498 cells.. Treatment of RCC cell lines with NVP-BEZ235 in vitro resulted in the nuclear translocation of p27, greater reduction in tumor cell proliferation, and more complete suppression of Akt, Mnk-1, eIF4E, and 4EBP-1 phosphorylation and cyclin D1 and hypoxia-inducible factor 2alpha (HIF2alpha) expression than that achieved with rapamycin. The reduction of HIF2alpha levels correlated with reduced HIF activity as determined by luciferase assay. NVP-BEZ235 induced growth arrest in both the 786-O and A498 xenografts that was associated with inhibition of Akt and S6 phosphorylation as well as the induction of apoptosis and reduction in markers of tumor cell proliferation. In contrast, rapamycin induced only minimal growth retardation.. Dual inhibition of PI3K/mTOR with NVP-BEZ235 induced growth arrest in RCC cell lines both in vitro and in vivo more effectively than inhibition of TORC1 alone. These results provide the rationale for the clinical assessment of agents such as NVP-BEZ235 in patients with advanced RCC.

Topics: Animals; Carcinoma, Renal Cell; Cell Death; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Enzyme Inhibitors; Female; Humans; Imidazoles; Kidney Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Multiprotein Complexes; Neoplasm Transplantation; Phosphoinositide-3 Kinase Inhibitors; Proteins; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

Glioblastoma, the most intractable cerebral tumor, is highly lethal. Recent studies suggest that cancer stem-like cells (CSLCs) have the capacity to repopulate tumors and mediate radio- and chemoresistance, implying that future therapies may need to turn from the elimination of rapidly dividing, but differentiated, tumor cells to specifically targeting the minority of tumor cells that repopulate the tumor. However, the mechanism by which glioblastoma CSLCs maintain their immature stem-like state or, alternatively, become committed to differentiation is poorly understood. Here, we show that the inactivation of mammalian target of rapamycin (mTor) by the mTor inhibitor rapamycin or knockdown of mTor reduced sphere formation and the expression of neural stem cell (NSC)/progenitor markers in CSLCs of the A172 glioblastoma cell line. Interestingly, combination treatment with rapamycin and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, not only reduced the expression of NSC/progenitor markers more efficiently than single-agent treatment, but also increased the expression of βIII-tubulin, a neuronal differentiation marker. Consistent with these results, a dual PI3K/mTor inhibitor, NVP-BEZ235, elicited a prodifferentiation effect on A172 CSLCs. Moreover, A172 CSLCs, which were induced to undergo differentiation by pretreatment with NVP-BEZ235, exhibited a significant decrease in their tumorigenicity when transplanted either subcutaneously or intracranially. Importantly, similar results were obtained when patient-derived glioblastoma CSLCs were used. These findings suggest that the PI3K/mTor signaling pathway is critical for the maintenance of glioblastoma CSLC properties, and targeting both mTor and PI3K of CSLCs may be an effective therapeutic strategy in glioblastoma.

Topics: Animals; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chromones; Drug Therapy, Combination; Enzyme Inhibitors; Glioblastoma; Humans; Imidazoles; Immunosuppressive Agents; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Morpholines; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Although the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway promotes survival signaling, inhibitors of PI3K and mTOR induce minimal cell death in PTEN (phosphatase and tensin homolog deleted from chromosome 10) mutant glioma. Here, we show that the dual PI3K-mTOR inhibitor PI-103 induces autophagy in a form of glioma that is resistant to therapy. Inhibitors of autophagosome maturation cooperated with PI-103 to induce apoptosis through the mitochondrial pathway, indicating that the cellular self-digestion process of autophagy acted as a survival signal in this setting. Not all inhibitors of mTOR synergized with inhibitors of autophagy. Rapamycin delivered alone induced autophagy, yet cells survived inhibition of autophagosome maturation because of rapamycin-mediated activation of Akt. In contrast, adenosine 5'-triphosphate-competitive inhibitors of mTOR stimulated autophagy more potently than did rapamycin, with inhibition of mTOR complexes 1 and 2 contributing independently to induction of autophagy. We show that combined inhibition of PI3K and mTOR, which activates autophagy without activating Akt, cooperated with inhibition of autophagy to cause glioma cells to undergo apoptosis. Moreover, the PI3K-mTOR inhibitor NVP-BEZ235, which is in clinical use, synergized with the lysosomotropic inhibitor of autophagy, chloroquine, another agent in clinical use, to induce apoptosis in glioma xenografts in vivo, providing a therapeutic approach potentially translatable to humans.

Topics: Animals; Autophagy; Cell Line, Tumor; Chloroquine; Drug Synergism; Flow Cytometry; Furans; Glioma; Histological Techniques; Humans; Imidazoles; Immunoblotting; Immunohistochemistry; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Mitochondria; Mutation; Oncogene Protein v-akt; Phosphoinositide-3 Kinase Inhibitors; PTEN Phosphohydrolase; Pyridines; Pyrimidines; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Transplantation, Heterologous

Mammalian target of rapamycin (mTOR) regulates cellular processes important for progression of human cancer. RAD001 (everolimus), an mTORC1 (mTOR/raptor) inhibitor, has broad antitumor activity in preclinical models and cancer patients. Although most tumor lines are RAD001 sensitive, some are not. Selective mTORC1 inhibition can elicit increased AKT S473 phosphorylation, involving insulin receptor substrate 1, which is suggested to potentially attenuate effects on tumor cell proliferation and viability. Rictor may also play a role because rictor kinase complexes (including mTOR/rictor) regulate AKT S473 phosphorylation. The role of raptor and rictor in the in vitro response of human cancer cells to RAD001 was investigated. Using a large panel of cell lines representing different tumor histotypes, the basal phosphorylation of AKT S473 and some AKT substrates was found to correlate with the antiproliferative response to RAD001. In contrast, increased AKT S473 phosphorylation induced by RAD001 did not correlate. Similar increases in AKT phosphorylation occurred following raptor depletion using siRNA. Strikingly, rictor down-regulation attenuated AKT S473 phosphorylation induced by mTORC1 inhibition. Further analyses showed no relationship between modulation of AKT phosphorylation on S473 and T308 and AKT substrate phosphorylation patterns. Using a dual pan-class I phosphatidylinositol 3-kinase/mTOR catalytic inhibitor (NVP-BEZ235), currently in phase I trials, concomitant targeting of these kinases inhibited AKT S473 phosphorylation, eliciting more profound cellular responses than mTORC1 inhibition alone. However, reduced cell viability could not be predicted from biochemical or cellular responses to mTORC1 inhibitors. These data could have implications for the clinical application of phosphatidylinositol 3-kinase/mTOR inhibitors.

Topics: Carrier Proteins; Cell Proliferation; Cell Survival; Everolimus; Humans; Imidazoles; Immunoblotting; Immunosuppressive Agents; Insulin Receptor Substrate Proteins; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinases; Proteins; Proto-Oncogene Proteins c-akt; Quinolines; Rapamycin-Insensitive Companion of mTOR Protein; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tumor Cells, Cultured

Phosphoinositide 3-kinase (PI3K)/protein kinase B/Akt and Ras/mitogen-activated protein kinase pathways are often constitutively activated in melanoma and have thus been considered as promising drug targets. Exposure of melanoma cells to NVP-BAG956, NVP-BBD130, and NVP-BEZ235, a series of novel, potent, and stable dual PI3K/mammalian target of rapamycin (mTOR) inhibitors, resulted in complete G1 growth arrest, reduction of cyclin D1, and increased levels of p27(KIP1), but negligible apoptosis. In contrast, treatment of melanoma with the pan-class I PI3K inhibitor ZSTK474 or the mTORC1 inhibitor rapamycin resulted only in minor reduction of cell proliferation. In a syngeneic B16 mouse melanoma tumor model, orally administered NVP-BBD130 and NVP-BEZ235 efficiently attenuated tumor growth at primary and lymph node metastatic sites with no obvious toxicity. Metastatic melanoma in inhibitor-treated mice displayed reduced numbers of proliferating and significantly smaller tumor cells. In addition, neovascularization was blocked and tumoral necrosis increased when compared with vehicle-treated mice. In conclusion, compounds targeting PI3K and mTOR simultaneously were advantageous to attenuate melanoma growth and they develop their potential by targeting tumor growth directly, and indirectly via their interference with angiogenesis. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential in metastatic melanoma therapy.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Administration, Oral; Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Cycle; Cell Proliferation; Drug Synergism; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; Imidazoles; Immunoblotting; Immunoenzyme Techniques; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Protein Transport; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Triazines; Tumor Cells, Cultured

Tuberous sclerosis (TSC) is a hamartoma syndrome in which renal and lung tumors cause the greatest morbidity. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1 and suppression of AKT. Recent studies indicate that inhibition of mTORC1 with RAD001 (everolimus) leads to rebound activation of AKT, which could protect tumors from drug-induced cell death. Here we examine the potential benefit of inhibition of both mTOR and AKT signaling in a mouse model of TSC, using a dual pan class I PI3K/mTOR catalytic small molecule inhibitor NVP-BEZ235.. Using ENU to enhance Tsc2+- kidney tumor development, both RAD001 (10 mg/kg PO 5 d/week) and NVP-BEZ235 (45 mg/kg PO QD) had equivalent effects in suppressing tumor development during a 4 week treatment period, with a 99% reduction in tumor cell mass. Marked reduction in activation of mTORC1, induction of cell cycle arrest, and absence of apoptotic cell death was seen in mice treated with either drug. However, when either was discontinued, there was prompt recovery of tumor growth, with extensive proliferation.. Both mTORC1 blockade alone and combined PI3K-mTOR blockade lead to suppression of tumor development but not tumor elimination in this TSC model.

Topics: Animals; Antineoplastic Agents; Carrier Proteins; Disease Models, Animal; Ethylnitrosourea; Everolimus; Imidazoles; Kidney Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiprotein Complexes; Neoplasm Recurrence, Local; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Proteins; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tuberous Sclerosis

Topics: Antineoplastic Agents; Breast Neoplasms; Female; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases