sirolimus and tanespimycin

Tuberous sclerosis complex (TSC) is a neurogenetic disorder that leads to elevated mechanistic targeting of rapamycin complex 1 (mTORC1) activity. Cilia can be affected by mTORC1 signaling, and ciliary deficits are associated with neurodevelopmental disorders. Here, we examine whether neuronal cilia are affected in TSC. We show that cortical tubers from TSC patients and mutant mouse brains have fewer cilia. Using high-content image-based assays, we demonstrate that mTORC1 activity inversely correlates with ciliation in TSC1/2-deficient neurons. To investigate the mechanistic relationship between mTORC1 and cilia, we perform a phenotypic screen for mTORC1 inhibitors with TSC1/2-deficient neurons. We identify inhibitors of the heat shock protein 90 (Hsp90) that suppress mTORC1 through regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Pharmacological inhibition of Hsp90 rescues ciliation through downregulation of Hsp27. Our study uncovers the heat-shock machinery as a druggable signaling node to restore mTORC1 activity and cilia due to loss of TSC1/2, and it provides broadly applicable platforms for studying TSC-related neuronal dysfunction.

Topics: Aging; Animals; Benzoquinones; Brain; Cilia; Down-Regulation; Heat-Shock Response; HSP27 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mechanistic Target of Rapamycin Complex 1; Mice, Knockout; Neurons; Phenotype; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Sirolimus; Time Factors; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Up-Regulation

Advanced prostate cancers that are resistant to all current therapies create a need for new therapeutic strategies. One recent innovative approach to cancer therapy is the simultaneous use of multiple FDA-approved drugs to target multiple pathways. A challenge for this approach is caused by the different solubility requirements of each individual drug, resulting in the need for a drug vehicle that is non-toxic and capable of carrying multiple water-insoluble antitumor drugs. Micelles have recently been shown to be new candidate drug solubilizers for anti cancer therapy.. This study set out to examine the potential use of multi-drug loaded micelles for prostate cancer treatment in preclinical models including cell line and mouse models for prostate cancers with Pten deletions. Specifically antimitotic agent docetaxel, mTOR inhibitor rapamycin, and HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin were incorporated into the micelle system (DR17) and tested for antitumor efficacy.. In vitro growth inhibition of prostate cancer cells was greater when all three drugs were used in combination compared to each individual drug, and packaging the drugs into micelles enhanced the cytotoxic effects. At the molecular level DR17 targeted simultaneously several molecular signaling axes important in prostate cancer including androgen receptor, mTOR, and PI3K/AKT. In a mouse genetic model of prostate cancer, DR17 treatment decreased prostate weight, which was achieved by both increasing caspase-dependent cell death and decreasing cell proliferation. Similar effects were also observed when DR17 was administered to nude mice bearing prostate cancer cells xenografts.. These results suggest that combining these three cancer drugs in multi-drug loaded micelles may be a promising strategy for prostate cancer therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzoquinones; Cell Line, Tumor; Cell Proliferation; Docetaxel; HSP90 Heat-Shock Proteins; Immunoblotting; Lactams, Macrocyclic; Male; Mice, Knockout; Mice, Transgenic; Micelles; Molecular Targeted Therapy; Neoplasms, Experimental; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; Taxoids; TOR Serine-Threonine Kinases

Despite clinical remission of epithelial ovarian cancer (EOC) after surgical resection and first-line chemotherapy, about 60% of patients will re-develop peritoneal metastasis and about 50% will relapse with chemoresistant disease. Clinical studies suggest that intra-peritoneal (i.p.) chemotherapy effectively treats residual EOC after cyto-reduction by gaining direct access into the peritoneal cavity, enabling elevated drug levels versus intravenous (i.v.) injection. However, chemoresistant disease is still problematic. To overcome resistance against microtubule stabilizing agents such as taxanes, epothilone B (EpoB) has merit, especially in combination with molecular targeted agents that inhibit heat shock protein 90 (Hsp90) and/or mammalian target of rapamycin (mTOR). In this paper, we report on the successful loading and solubilization of EpoB in a poly(d,l-lactic-co-glycolic acid)-block-poly(ethylene glycol)-block-poly(d,l-lactic-co-glycolic acid) (PLGA-b-PEG-b-PLGA) thermosensitive gel (g-E). Further, we report on successful co-loading of 17-AAG (Hsp90) and rapamycin (mTOR) (g-EAR). After i.p. injection in mice, g-EAR showed gelation in the peritoneum and sustained, local-regional release of EpoB, 17-AAG, and rapamycin. In a luciferase-expressing ES-2 (ES-2-luc) ovarian cancer xenograft model, single i.p. injections of g-E and g-EAR delayed bioluminescence from metastasizing ES-2-luc cells for 2 and 3weeks, respectively, despite fast drug release for g-EAR in vivo versus in vitro. In summary, a PLGA-b-PEG-b-PLGA sol-gel has loading and release capacities for EpoB and its combinations with 17-AAG and rapamycin, enabling a platform for i.p. delivery, sustained multi-drug exposure, and potent antitumor efficacy in an ES-2-luc, ovarian cancer i.p. xenograft model.

Topics: Animals; Antineoplastic Agents; Benzoquinones; Cell Line, Tumor; Drug Liberation; Epothilones; Female; Gels; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mice, Nude; Ovarian Neoplasms; Polyesters; Polyethylene Glycols; Sirolimus; TOR Serine-Threonine Kinases

Epothilones are microtubule inhibitors that are promising alternatives to paclitaxel due to enhanced anticancer efficacy. While epothilones are slightly more water soluble than paclitaxel and more active against paclitaxel-resistant cells, they still require formulation with Cremophor EL and/or cosolvents and drug resistance still limits therapeutic efficacy. In this report, we showed that the combinational treatment of epothilone B (EpoB), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG, Hsp90 inhibitor), and rapamycin (mTOR inhibitor) displays strong anticancer activity in vitro and in vivo. To address the poor water solubility of this 3 drug-combination, they were co-loaded into poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles, and the 3-in-1 loaded PEG-b-PLA micelle (m-EAR) was characterized in terms of drug loading efficiency, particle size, release kinetics. The m-EAR achieved high levels of all three drugs in water; formed micelles with hydrodynamic diameters at ca. 30nm and released the drugs in a sustained manner in vitro at rates slower than individually loaded PEG-b-PLA micelles. In A549-derived xenograft mice, m-EAR (2.0, 15.0, and 7.5mg/kg) caused tumor regression after four weekly injections, whereas EpoB alone (2.0mg/kg) was the same as control. No severe changes in body weight relative to PBS control were observed, attesting to the safety of m-EAR. Collectively, these results suggest that m-EAR provides a simple, but effective and safe EpoB-based combination nanomedicine for cancer therapy.

Topics: A549 Cells; Animals; Antineoplastic Agents; Benzoquinones; Cell Survival; Drug Combinations; Drug Liberation; Epothilones; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mice, Nude; Micelles; Neoplasms; Polyethylene Glycols; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden

Objective：To explore the influence of co-inhibiting mTORC2 and HSP90 on the proliferation and apoptosis of multiple myeloma(MM) cell line U266.. During culture, the human MM cell line U266 were treated with 20 nmol/L of rapamycin, 600 nmol/L 17-AAG, 20 nmol/L of rapamycin + 600 nmol/L 17-AGG and phosphate-buffered saline (PBS), then the growth inhibition rate, morphologic changes, apoptosis rate and the expression of caspase 3 and ATK protein in U266 cells were compared and analyzed.. The rapamycin and 17-AAG both could inhibit the growth of U266 cells, while the inhibitory effect of rapamycin in combination with 17-AAG on growth of U266 cells was significantly higher them that of rapamycin and 17-AAG alone and control (PBS); the apoptosis rate of U266 cells treated with rapamycin, 17-AAG and their combination was higher than that of control PBS groups, and the efficacy of 2 drug conbination was higher than that of control PBS group, and the efficacy of 2 drug combination was superior to single drug. The expression levels of caspase 3 and ATK in U266 cells treated with rapamycin, 17-AAG and their combination were higher and lower than those in control group respectively, and the efficacy of 2 drug combination was superior to signle drug. There were significant difference between them (P<0.05).. The co-inhibition of mTORC2 and HSP90 can suppress the proliferation and induce the apoptosis of MM cells.

Topics: Apoptosis; Benzoquinones; Caspase 3; Cell Line, Tumor; Cell Proliferation; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mechanistic Target of Rapamycin Complex 2; Multiple Myeloma; Multiprotein Complexes; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals from growth factors, nutrients, and cellular energy status to control a wide range of metabolic processes, including mRNA biogenesis; protein, nucleotide, and lipid synthesis; and autophagy. Deregulation of the mTORC1 pathway is found in cancer as well as genetic disorders such as tuberous sclerosis complex (TSC) and sporadic lymphangioleiomyomatosis. Recent studies have shown that the mTORC1 inhibitor rapamycin and its analogs generally suppress proliferation rather than induce apoptosis. Therefore, it is critical to use alternative strategies to induce death of cells with activated mTORC1. In this study, a small-molecule screen has revealed that the combination of glutaminase (GLS) and heat shock protein 90 (Hsp90) inhibitors selectively triggers death of TSC2-deficient cells. At a mechanistic level, high mTORC1-driven translation rates in TSC1/2-deficient cells, unlike wild-type cells, sensitizes these cells to endoplasmic reticulum (ER) stress. Thus, Hsp90 inhibition drives accumulation of unfolded protein and ER stress. When combining proteotoxic stress with oxidative stress by depletion of the intracellular antioxidant glutathione by GLS inhibition, acute cell death is observed in cells with activated mTORC1 signaling. This study suggests that this combination strategy may have the potential to be developed into a therapeutic use for the treatment of mTORC1-driven tumors.

Topics: Animals; Apoptosis; Benzoquinones; Cell Line, Tumor; Cell Shape; Cell Survival; Glutamate Dehydrogenase; Glutaminase; Glutamine; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mechanistic Target of Rapamycin Complex 1; Mice; Models, Biological; Multiprotein Complexes; Oxidation-Reduction; Phenotype; Sirolimus; Small Molecule Libraries; Sulfides; Thiadiazoles; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Traumatic brain injury (TBI) is caused by both primary and secondary injury mechanisms, all of which cause neuronal cell death and functional deficits. Both apoptosis and autophagy participated in neuronal cell death and functional loss induced following TBI. Preclinical findings implicate that 17-allylamino-demethoxygeldanamycin (17-AAG), an anticancer drug in clinical, present neuroprotection actions in multiple neurological disorders, but whether 17-AAG is capable of modulating neuronal autophagy has never been addressed. The present study was designed to determine the hypothesis that17-AAG treatment could confer neuroprotection in a rat model of TBI. We also used an autophagy inhibitor 3-methyladenine (3-MA) as well as an autophagy inducer rapamycin (RAPA) to test its underlining mechanisms. Our results showed that post-TBI administration of 17-AAG could attenuate brain edema, decrease neuronal death, as well as improve the recovery of motor function. Afterwards, in our model, 17-AAG treatment protected against TBI-induced apoptosis activation as well as enhanced neuronal autophagy. The present study provides novel clues in understanding the mechanisms of which 17-AAG exerts its neuroprotective activity on neurological disorders.

Topics: Adenine; Animals; Apoptosis; Autophagy; Benzoquinones; Brain Edema; Brain Injuries; Cell Survival; Cerebral Cortex; Female; Lactams, Macrocyclic; Motor Skills; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley; Sirolimus

To explore apoptosis of multiple myeloma (MM) cells and its mechanism by the combined inhibition of mTORC2 signaling pathway and heat shock protein 90.. The effects of Rapamycin, 17-AAG and the combination on proliferation of MM cell lines U266 and KM3 were assessed using MTT at different time points (0, 8, 24, 48 hour). Cell apoptosis and cell cycle distribution were measured by flow cytometry. The specific proteins p-AKT (ser473), p-AKT (thr450), p-S6 (S235/236) and AKT were detected by Western blotting.. Rapamycin, 17- AAG and the combination suppressed the proliferation of MM cell lines U266 and KM3, especially the combination of Rapamycin and 17-AAG synergistically inhibited the proliferation (P<0.05); Rapamycin induced G1 arrest both at 24 and 48 hours, 17-AAG also induced G1 arrest, especially at 48 hours (P<0.01); Rapamycin, 17-AAG alone decreased the expression of AKT and induced MM cell apoptosis to some extent (P<0.01); Chronic rapamycin treatment inhibited mTORC2; Inhibition of both mTORC2 and chaper on pathways degraded AKT and induced MM cell apoptosis, which was significantly higher than that of any single agent (P<0.01).. Inhibition of both mTORC2 and chaper on pathways decreased the expression of AKT to induce apoptosis of MM cells in vitro.

Topics: Apoptosis; Benzoquinones; Cell Cycle; Cell Division; Cell Line, Tumor; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mechanistic Target of Rapamycin Complex 2; Multiple Myeloma; Multiprotein Complexes; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

A current treatment strategy for peritoneal ovarian cancer is a combination of peritoneal surgery and multi-drug-based chemotherapy that often involves intraperitoneal (IP) injection. A thermosensitive poly-(D,L-lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly-(D,L-lactide-co-glycolide) (PLGA-b-PEG-b-PLGA) hydrogel platform (thermogels) enabled gel loading of poorly work-soluble paclitaxel (cytotoxic agent), 17-allylamino-17-demethoxygeldanamycin (17-AAG, heat shock protein inhibitor), and rapamycin (mammalian target of rapamycin protein inhibitor). PLGA-b-PEG-b-PLGA thermogels (15%) carrying paclitaxel, 17-AAG, and rapamycin (named Triogel) made a successful transition from a free-flowing solution below ambient temperature to a gel depot at body temperature. Triogel gradually released paclitaxel, 17-AAG, and rapamycin at an equal release rate in response to the physical gel erosion. In an ES-2-luc ovarian cancer xenograft model, a single IP injection of Triogel (60, 60, and 30 mg/kg of paclitaxel, 17-AAG, and rapamycin, respectively) significantly reduced tumor burden and prolonged survival of ES-2-luc-bearing nude mice without notable systemic toxicity relative to those delivered by poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles in solution via IP or intravenous (IV) injection route. These results show a great potential of a biodegradable thermogel platform carrying multi-drugs for IP chemotherapy in peritoneal ovarian cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzoquinones; Cell Line, Tumor; Cell Survival; Drug Carriers; Drug Combinations; Drug Liberation; Female; Humans; Hydrogels; Injections, Intraperitoneal; Injections, Intravenous; Lactams, Macrocyclic; Mice, Nude; Molecular Structure; Paclitaxel; Phase Transition; Polyesters; Polyethylene Glycols; Sirolimus; Transition Temperature; Xenograft Model Antitumor Assays

Constitutive activation of Akt is believed to be an oncogenic signal in multiple myeloma and is associated with poor patient prognosis and resistance to available treatment. The stability of Akt proteins is regulated by phosphorylating the highly conserved turn motif (TM) of these proteins and the chaperone protein HSP90. In this study we investigate the antitumor effects of inhibiting mTORC2 plus HSP90 in myeloma cell lines. We show that chronic exposure of cells to rapamycin can inhibit mTORC2 pathway, and AKT will be destabilized by administration of the HSP90 inhibitor 17-allylamino-geldanamycin (17-AAG). Finally, we show that the rapamycin synergizes with 17-AAG and inhibits myeloma cells growth and promotes cell death to a greater extent than either drug alone. Our studies provide a clinical rationale of use mTOR inhibitors and chaperone protein inhibitors in combination regimens for the treatment of human blood cancers.

Topics: Benzoquinones; Cell Death; Cell Line, Tumor; Enzyme Stability; Humans; Lactams, Macrocyclic; Mechanistic Target of Rapamycin Complex 2; Multiple Myeloma; Multiprotein Complexes; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Though prostate cancer (PCa) has slow progression, the hormone refractory (HRCP) and metastatic entities are substantially lethal and lack effective treatments. Transcription factor Slug is critical in regulating metastases of various tumors including PCa. Here we studied targeted therapy against Slug using combination of 3 drugs targeting 3 pathways respectively converging via Slug and further regulating PCa metastasis. Using in vitro assays we confirmed that Slug up-regulation incurred inhibition of E-cadherin that was anti-metastatic, and inhibited Bim-regulated cell apoptosis in PCa. Upstream PTEN/Akt, mTOR, Erk, and AR/Hsp90 pathways were responsible for Slug up-regulation and each of these could be targeted by rapamycin, CI-1040, and 17-AAG respectively. In 4 PCa cell lines with different traits in terms of PTEN loss and androgen sensitivity we tested the efficacy of mono- and combined therapy with the drugs. We found that metastatic capacity of the cells was maximally inhibited only when all 3 drugs were combined, due to the crosstalk between the pathways. 17-AAG decreases Slug expression via blockade of HSP90-dependent AR stability. Combination of rapamycin and CI-1040 diminishes invasiveness more potently in PCa cells that are androgen insensitive and with PTEN loss. Slug inhibited Bim-mediated apoptosis that could be rescued by mTOR/Erk/HSP90 inhibitors. Using mouse models for circulating PCa DNA quantification, we found that combination of mTOR/Erk/HSP90 inhibitors reduced circulating PCa cells in vivo significantly more potently than combination of 2 or monotherapy. Conclusively, combination of mTOR/Erk/Hsp90 inhibits metastatic capacity of prostate cancer via Slug inhibition.

Topics: Androgens; Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Benzoquinones; Cell Line, Tumor; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Male; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Prostate; Prostatic Neoplasms; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Snail Family Transcription Factors; Transcription Factors

Cotylenin A, a plant growth regulator, and rapa-mycin, an inhibitor of the mammalian target of rapamycin, are potent inducers of differentiation in myeloid leukemia cells and also synergistically inhibit the proliferation of several human breast cancer cell lines including MCF-7 in vitro and in vivo. However, the mechanisms of the combined effects of cotylenin A and rapamycin are still unknown. Activated Akt induced by rapamycin has been suggested to attenuate the growth-inhibitory effects of rapamycin, serving as a negative feedback mechanism. In this study, we found that cotylenin A could suppress rapamycin-induced phosphorylation of Akt (Ser473) in MCF-7 cells and lung carcinoma A549 cells and that cotylenin A also enhanced the rapamycin-induced growth inhibition of MCF-7 and A549 cells. ISIR-005 (a synthetic cotylenin A-derivative) was able to enhance rapamycin‑induced growth inhibition and could also markedly inhibit rapamycin-induced phosphorylation of Akt. We also found that the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) or arsenic trioxide (ATO) in combination with rapamycin markedly inhibited the growth of MCF-7 cells and 17-AAG or ATO suppressed rapamycin-induced phosphorylation of Akt. The PI3K inhibitor LY294002 also suppressed rapamycin-induced phosphorylation of Akt and combined treatment showed synergistic growth inhibition of MCF-7 cells. Rapamycin inhibited growth more significantly in Akt siRNA-transfected MCF-7 cells than in control siRNA-transfected MCF-7 cells. These results suggest that the inhibition of rapamycin-induced Akt phosphorylation by cotylenin A correlates with their effective growth inhibition of cancer cells.

Topics: Benzoquinones; Breast Neoplasms; Cell Proliferation; Chromones; Diterpenes; Drug Synergism; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; MCF-7 Cells; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Sirolimus

Concurrent delivery of multiple poorly water-soluble anticancer drugs has been a great challenge due to the toxicities exerted by different surfactants or organic solvents used in solubilizing individual drugs. We previously found that poly(ethylene glycol)-block-poly(D, L-lactic acid) (PEG-b-PLA) micelles can serve as a safe delivery platform for simultaneous delivery of paclitaxel (PTX), 17-allylamino-17-demethoxygeldanamycin (17-AAG), and rapamycin (RAP) to mice. The high tolerance of this polymeric micelle formulation by mice allowed us to investigate the pharmacokinetics of the 3 co-delivered drugs. In this study, it was shown that 3-in-1 PEG-b-PLA micelle delivering high doses of PTX, 17-AAG, and RAP (60, 60, and 30 mg/kg, respectively) significantly increased the values of the area under the plasma concentration-time curves (AUC) of PTX and RAP in mice compared to the drugs delivered individually, while the pharmacokinetic parameters of 17-AAG were similar in both 3-in-1 and single drug-loaded PEG-b-PLA micelle formulations. Moreover, pharmacokinetic study using 2-in-1 micelles indicated that the augmented AUC value of RAP was due to the co-delivery of 17-AAG, while the increase in AUC of PTX was more likely caused by the co-delivery of RAP. In contrast, when 3-in-1 and single drug-loaded PEG-b-PLA micelles were administrated at modest dose (PTX, 17-AAG, and RAP at 10, 10, and 5 mg/kg, respectively), pharmacokinetic differences of individual drugs between 3-in-1 and single drug formulations were eliminated. These results suggest that 3-in-1 PEG-b-PLA micelles can concurrently deliver PTX, 17-AAG, and RAP without changing the pharmacokinetics of each drug at modest doses, but altered pharmacokinetic profiles emerge when drugs are delivered at higher doses.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Benzoquinones; Dose-Response Relationship, Drug; Drug Carriers; Drug Combinations; Female; Lactams, Macrocyclic; Lactates; Mice; Micelles; Paclitaxel; Polyethylene Glycols; Sirolimus

Constitutive activation of the kinases Akt or protein kinase C (PKC) in blood cancers promotes tumor-cell proliferation and survival and is associated with poor patient survival. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) regulates the stability of Akt and conventional PKC (cPKC; PKCα and PKCβ) proteins by phosphorylating the highly conserved turn motif of these proteins. In cells that lack mTORC2 function, the turn motif phosphorylation of Akt and cPKC is abolished and therefore Akt and cPKC protein stability is impaired. However, the chaperone protein HSP90 can stabilize Akt and cPKC, partially rescuing the expression of these proteins. In the present study, we investigated the antitumor effects of inhibiting mTORC2 plus HSP90 in mouse and human leukemia cell models and show that the HSP90 inhibitor 17-allylaminogeldanamycin (17-AAG) preferentially inhibits Akt and cPKC expression and promotes cell death in mTORC2 deficient pre-B leukemia cells. Furthermore, we show that 17-AAG selectively inhibits mTORC2 deficient leukemia cell growth in vivo. Finally, we show that the mTOR inhibitors rapamycin and pp242 work together with 17-AAG to inhibit leukemia cell growth to a greater extent than either drug alone. These studies provide a mechanistic and clinical rationale to combine mTOR inhibitors with chaperone protein inhibitors to treat human blood cancers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzoquinones; Cells, Cultured; Drug Evaluation, Preclinical; HEK293 Cells; HSP90 Heat-Shock Proteins; Humans; Indoles; Jurkat Cells; Lactams, Macrocyclic; Leukemia; Mice; Mice, Transgenic; Molecular Chaperones; Purines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Trans-Activators; Transcription Factors

Triolimus is a first-in-class, multidrug-loaded micelle containing paclitaxel, rapamycin, and 17-AAG. In this study, we examine the antitumor mechanisms of action, efficacy, and toxicity of Triolimus in vitro and in vivo. In vitro cytotoxicity testing of Triolimus was conducted using two aggressive adenocarcinomas including the lung cancer cell line, A549, and breast cancer cell line, MDA-MB-231. The three-drug combination of paclitaxel, rapamycin, and 17-AAG displayed potent cytotoxic synergy in both A549 and MDA-MB-231 cell lines. Mechanistically, the drug combination inhibited both the Ras/Raf/mitogen-activated protein kinase and PI3K/Akt/mTOR pathways. Triolimus was advanced into tumor xenograft models for assessment of efficacy, toxicity, and mechanisms of action. In vivo, a three-infusion schedule of Triolimus inhibited A549 and MDA-MB-231 tumor growth far more potently than paclitaxel-containing micelles and effected tumor cures in MDA-MB-231 tumor-bearing animals. Tumor growth delays resulted from a doubling in tumor cell apoptosis and a 50% reduction in tumor cell proliferation compared with paclitaxel-containing micelles. Enhanced antitumor efficacy was achieved without clinically significant increases in acute toxicity. Thus, Triolimus displays potent synergistic activity in vitro and antitumor activity in vivo with comparable toxicity to paclitaxel. These observations provide strong support for further development of Triolimus and an important proof of concept for safe, effective nanoparticle-based delivery of three complementary anticancer agents.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzoquinones; Cell Death; Cell Line, Tumor; Cell Proliferation; Drug Combinations; Drug Synergism; Humans; Lactams, Macrocyclic; Mice; Mice, Nude; Micelles; Paclitaxel; Signal Transduction; Sirolimus; Xenograft Model Antitumor Assays

Poly(ethylene glycol)-block-poly(D,L-lactic acid) (PEG-b-PLA) micelles have a proven capacity for drug solubilization and have entered phase III clinical trials as a substitute for Cremophor EL in the delivery of paclitaxel in cancer therapy. PEG-b-PLA is less toxic than Cremophor EL, enabling a doubling of paclitaxel dose in clinical trials. We show that PEG-b-PLA micelles act as a 3-in-1 nanocontainer for paclitaxel, 17-allylamino-17-demethoxygeldanamycin (17-AAG), and rapamycin for multiple drug solubilization. 3-in-1 PEG-b-PLA micelles were ca. 40 nm in diameter; dissolved paclitaxel, 17-AAG, and rapamycin in water at 9.0 mg/mL; and were stable for 24 h at 25 °C. The half-life for in vitro drug release (t(1/2)) for 3-in-1 PEG-b-PLA micelles was 1-15 h under sink conditions and increased in the order of 17-AAG, paclitaxel, and rapamycin. The t(1/2) values correlated with log P(o/w) values, implicating a diffusion-controlled mechanism for drug release. The IC(50) value of 3-in-1 PEG-b-PLA micelles for MCF-7 and 4T1 breast cancer cell lines was 114 ± 10 and 25 ± 1 nM, respectively; combination index (CI) analysis showed that 3-in-1 PEG-b-PLA micelles exert strong synergy in MCF-7 and 4T1 breast cancer cell lines. Notably, concurrent intravenous (iv) injection of paclitaxel, 17-AAG, and rapamycin using 3-in-1 PEG-b-PLA micelles was well-tolerated by FVB albino mice. Collectively, these results suggest that PEG-b-PLA micelles carrying paclitaxel, 17-AAG, and rapamycin will provide a simple yet safe and efficacious 3-in-1 nanomedicine for cancer therapy.

Topics: Benzoquinones; Cell Line, Tumor; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lactic Acid; Micelles; Models, Theoretical; Paclitaxel; Polyesters; Polyethylene Glycols; Polymers; Sirolimus; Solubility; TOR Serine-Threonine Kinases; Water

Ras-driven tumors are often refractory to conventional therapies. Here we identify a promising targeted therapeutic strategy for two Ras-driven cancers: Nf1-deficient malignancies and Kras/p53 mutant lung cancer. We show that agents that enhance proteotoxic stress, including the HSP90 inhibitor IPI-504, induce tumor regression in aggressive mouse models, but only when combined with rapamycin. These agents synergize by promoting irresolvable ER stress, resulting in catastrophic ER and mitochondrial damage. This process is fueled by oxidative stress, which is caused by IPI-504-dependent production of reactive oxygen species, and the rapamycin-dependent suppression of glutathione, an important endogenous antioxidant. Notably, the mechanism by which these agents cooperate reveals a therapeutic paradigm that can be expanded to develop additional combinations.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzoquinones; Carcinoma, Non-Small-Cell Lung; eIF-2 Kinase; Endoplasmic Reticulum; Fluorescent Antibody Technique; Glutathione; HSP90 Heat-Shock Proteins; In Situ Nick-End Labeling; Lactams, Macrocyclic; Mice; Mitochondria; Molecular Targeted Therapy; Nerve Sheath Neoplasms; Oxidative Stress; Polymerase Chain Reaction; Proto-Oncogene Proteins p21(ras); ras Proteins; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Sirolimus; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Acquisition of mesenchymal phenotype by epithelial cells by means of epithelial-mesenchymal transition (EMT) is considered as an early event in the multistep process of tumor metastasis. Therefore, inhibition of EMT might be a rational strategy to prevent metastasis.. Using the global gene expression profile from a cell culture model of transforming growth factor-β (TGF-β)-induced EMT, we identified potential EMT inhibitors. We used a publicly available database (www.broad.mit.edu/cmap) comprising gene expression profiles obtained from multiple different cell lines in response to various drugs to derive negative correlations to EMT gene expression profile using Connectivity Map, a pattern matching tool.. Experimental validation of the identified compounds showed rapamycin as a novel inhibitor of TGF-β signaling along with 17-AAG, a known modulator of TGF-β pathway. Both of these compounds completely blocked EMT and the associated migratory and invasive phenotype. The other identified compound, LY294002, demonstrated a selective inhibition of mesenchymal markers, cell migration and invasion, without affecting the loss of E-cadherin expression or Smad phosphorylation.. Our data reveal that rapamycin is a novel modulator of TGF-β signaling, and along with 17-AAG and LY294002, could be used as therapeutic agent for inhibiting EMT. This study demonstrates the potential of a systems approach in identifying novel modulators of a complex biological process.

Topics: Adenocarcinoma; Adenocarcinoma, Bronchiolo-Alveolar; Benzoquinones; Biomarkers, Tumor; Blotting, Western; Cadherins; Cell Movement; Chromones; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Gene Expression Profiling; HSP90 Heat-Shock Proteins; Humans; Immunosuppressive Agents; Lactams, Macrocyclic; Lung Neoplasms; Morpholines; Oligonucleotide Array Sequence Analysis; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Sirolimus; Smad Proteins; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Osteosarcoma is highly resistant to current chemotherapy regimens. Novel therapeutic approaches, potentially involving targeting of specific survival pathways, are needed. We used 17-AAG to inhibit Hsp90 and rapamycin to inhibit mTOR, in the osteosarcoma cell lines, HOS and KHOS/NP. HOS and KHOS cells were treated for 24 and 48 h with 17-AAG or rapamycin and studied drug-induced apoptosis, cell cycle, mitochondrial membrane potential and levels of reduced glutathione (GSH), dephosphorylation of signal transduction proteins in the Akt/MAP kinase pathway and mTOR signaling. 17-AAG was a potent inducer of apoptosis, involving effective depletion of GSH and mitochondrial membrane (MM) depolarization, strong activation of caspase-8 and -9 and release of AIF from mitochondria to the cytosol. Furthermore, 17-AAG down-regulated pAkt, p44Erk, p-mTOR, p70S6, TSC1/2 and pGSK-3beta. Treatment with 17-AAG also caused down-regulation of cyclin D1, GADD45a, GADD34 and pCdc2 and upregulation of cyclin B1 and mitotic block. A decrease in Hsp90 and increase in Hsp70 and Hsp70 C-terminal fragments were also observed. Rapamycin was a less potent inducer of apoptosis, involving a small decrease in GSH and MM potential with no activation of caspases or release of AIF. Rapamycin strongly inhibited cell growth with an increase in G1 and a decrease in S-phase of the cell cycle concomitant with down-regulation of cyclin D1. Rapamycin also down-regulated the activity of p70S6, pAkt and p-mTOR, but had no effect on pGSK-3beta, p44Erk, pCdc2, TSC1/2 or Hsp70 or Hsp90. We conclude that Hsp90 inhibition merits further study in the therapy of osteosarcoma.

Topics: Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Bone Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Humans; Lactams, Macrocyclic; Mitochondrial Membranes; Osteosarcoma; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

High-frequency microsatellite-instable (MSI-H) tumors account for approximately 15% of colorectal cancers. Therapeutic decisions for colorectal cancer are empirically based and currently do not emphasize molecular subclassification despite an increasing collection of gene expression information. Our objective was to identify low molecular weight compounds with preferential activity against MSI colorectal cancers using combined gene expression data sets.. Three expression/query signatures (discovery data set) characterizing MSI-H colorectal cancer were matched with information derived from changes induced in cell lines by 164 compounds using the systems biology tool "Connectivity Map." A series of sequential filtering and ranking algorithms were used to select the candidate compounds. Compounds were validated using two additional expression/query signatures (validation data set). Cytotoxic, cell cycle, and apoptosis effects of validated compounds were evaluated in a panel of cell lines.. Fourteen of the 164 compounds were validated as targeting MSI-H cell lines using the bioinformatics approach; rapamycin, LY-294002, 17-(allylamino)-17-demethoxygeldanamycin, and trichostatin A were the most robust candidate compounds. In vitro results showed that MSI-H cell lines due to hypermethylation of MLH1 are preferentially targeted by rapamycin (18.3 versus 4.4 mumol/L; P = 0.0824) and LY-294002 (15.02 versus 10.37 mumol/L; P = 0.0385) when compared with microsatellite-stable cells. Preferential activity was also observed in MSH2 and MSH6 mutant cells.. Our study shows that the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway is of special relevance in mismatch repair-deficient colorectal cancer. In addition, we show that amalgamation of gene expression information across studies provides a robust approach for selection of potential therapies corresponding to specific groups of patients.

Topics: Algorithms; Antineoplastic Agents; Benzoquinones; Cell Cycle; Cell Line, Tumor; Chromones; Colorectal Neoplasms; Computational Biology; DNA Mismatch Repair; Drug Evaluation, Preclinical; Enzyme Inhibitors; Gene Expression Profiling; Humans; Hydroxamic Acids; Immunosuppressive Agents; Lactams, Macrocyclic; Microsatellite Instability; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sirolimus

Increased Akt phosphorylation was reported in cancer cell lines and tumor tissues of patients exposed to rapamycin, a response likely contributing to the attenuated antitumor activity of rapamycin. It is, therefore, necessary to develop and validate combination strategies to reverse rapamycin-induced Akt signaling. We now report that Akt activation in response to rapamycin is abrogated by 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor. Rapamycin/17-AAG combination results in an enhanced antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cells. In combination 17-AAG confers potent suppression of Raf-MEK-extracellular signal-regulated kinase signaling, a pathway that is otherwise not inhibited by rapamycin individually. Importantly, 17-AAG cooperates with rapamycin to block the phosphorylation of the mammalian target of rapamycin at Ser2448, as well as its downstream effectors ribosomal p70 S6 kinase and eukaryotic initiation factor 4E binding protein 1, which is accompanied by a substantial reduction in cyclins D1 and E. The potency of rapamycin/17-AAG combination is not affected by the activation of insulin-like growth factor 1 receptor signaling, which has been previously shown to diminish the antiproliferative activity of rapamycin. Rapamycin/17-AAG combination alleviates the induction of HSP90 protein, a heat shock response frequently associated with 17-AAG monotherapy. Our findings establish a mechanistic rationale for a combination approach using rapamycin and 17-AAG in the treatment of breast cancer.

Topics: Benzoquinones; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Extracellular Signal-Regulated MAP Kinases; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Protein Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway and the heat shock protein family are up-regulated in multiple myeloma and are both regulators of the cyclin D/retinoblastoma pathway, a critical pathway in multiple myeloma. Inhibitors of mTOR and HSP90 protein have showed in vitro and in vivo single-agent activity in multiple myeloma. Our objective was to determine the effects of the mTOR inhibitor rapamycin and the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on multiple myeloma cells.. Multiple myeloma cell lines were incubated with rapamycin (0.1-100 nmol/L) and 17-AAG (100-600 nmol/L) alone and in combination.. In this study, we showed that the combination of rapamycin and 17-AAG synergistically inhibited proliferation, induced apoptosis and cell cycle arrest, induced cleavage of poly(ADP-ribose) polymerase and caspase-8/caspase-9, and dysregulated signaling in the phosphatidylinositol 3-kinase/AKT/mTOR and cyclin D1/retinoblastoma pathways. In addition, we showed that both 17-AAG and rapamycin inhibited angiogenesis and osteoclast formation, indicating that these agents target not only multiple myeloma cells but also the bone marrow microenvironment.. These studies provide the basis for potential clinical evaluation of this combination for multiple myeloma patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzoquinones; Bone Marrow Cells; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Drug Synergism; HSP90 Heat-Shock Proteins; Humans; Intercellular Signaling Peptides and Proteins; Lactams, Macrocyclic; Models, Biological; Multiple Myeloma; Neovascularization, Physiologic; Osteoclasts; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Animals; Antineoplastic Agents; Benzamides; Benzoquinones; Cell Cycle Proteins; DNA-Binding Proteins; E2F Transcription Factors; Gefitinib; Genes, Tumor Suppressor; HSP90 Heat-Shock Proteins; Humans; Imatinib Mesylate; Indoles; Lactams, Macrocyclic; Mutation; Neoplasms; Phosphoric Monoester Hydrolases; Piperazines; PTEN Phosphohydrolase; Pyrimidines; Pyrroles; Quinazolines; Rifabutin; Signal Transduction; Sirolimus; Transcription Factors; Tumor Suppressor Proteins