ABSTRACT
Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.
Subject(s)
Gene Expression Regulation, Neoplastic/physiology , HSP90 Heat-Shock Proteins/metabolism , Neoplasms/metabolism , Proteomics/methods , Animals , Antineoplastic Agents/pharmacology , Benzodioxoles/pharmacology , Cell Line, Tumor , Computational Biology , Drug Discovery , Gene Expression Regulation, Neoplastic/drug effects , HSP90 Heat-Shock Proteins/genetics , Humans , Neoplasms/genetics , Purines/pharmacology , Signal TransductionABSTRACT
Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Because of the absence of identified targets and targeted therapies, and due to a heterogeneous molecular presentation, treatment guidelines for patients with TNBC include only conventional chemotherapy. Such treatment, while effective for some, leaves others with high rates of early relapse and is not curative for any patient with metastatic disease. Here, we demonstrate that these tumors are sensitive to the heat shock protein 90 (Hsp90) inhibitor PU-H71. Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with this agent. Notably, TNBC tumors respond to retreatment with PU-H71 for several cycles extending for over 5 months without evidence of resistance or toxicity. Through a proteomics approach, we show that multiple oncoproteins involved in tumor proliferation, survival, and invasive potential are in complex with PU-H71-bound Hsp90 in TNBC. PU-H71 induces efficient and sustained downregulation and inactivation, both in vitro and in vivo, of these proteins. Among them, we identify downregulation of components of the Ras/Raf/MAPK pathway and G(2)-M phase to contribute to its anti-proliferative effect, degradation of activated Akt and Bcl-xL to induce apoptosis, and inhibition of activated NF-kappaB, Akt, ERK2, Tyk2, and PKC to reduce TNBC invasive potential. The results identify Hsp90 as a critical and multimodal target in this most difficult to treat breast cancer subtype and support the use of the Hsp90 inhibitor PU-H71 for clinical trials involving patients with TNBC.
Subject(s)
Benzodioxoles/pharmacology , Breast Neoplasms/drug therapy , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Purines/pharmacology , Antineoplastic Agents/pharmacology , Benzodioxoles/therapeutic use , Cell Line, Tumor , Female , Humans , Multiprotein Complexes/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Purines/therapeutic use , Receptor, ErbB-2/deficiency , Receptors, Estrogen/deficiency , Receptors, Progesterone/deficiency , Remission InductionABSTRACT
A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated among these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors.
Subject(s)
Drug Design , HSP90 Heat-Shock Proteins/drug effects , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding, Competitive , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , Molecular Probes/chemical synthesisABSTRACT
Heat shock protein 90 (Hsp90) is a molecular chaperone that has emerged as an important target in cancer and several other diseases, such as neurodegenerative diseases, nerve injuries, inflammation, and infection. Discovery of novel agents that inhibit Hsp90 and have druglike properties is therefore a major focus in several academic and industrial laboratories. In this study, the authors describe the development and optimization in a 384-well format of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization, which measures competitive binding of red-shifted fluorescently labeled geldanamycin (GM-cy3B) to Hsp90 found in the NCI-N417 small-cell lung carcinoma cells. The authors demonstrate that GMcy3B binds with high affinity and specificity to cellular Hsp90. The assay results in excellent signal-to-noise ratios (>10) and Z' values (>0.75) at tracer concentrations greater than 4 nM and 1 microg/well of total NCI-N417 protein, indicating a robust assay. It also equilibrates after 5 h of incubation at room temperature and remains stable for up to 24 h. Furthermore, it is a simple mix-and-read format that is cost-effective and uses only low amounts of fluorophore and cell lysates. A study using more than 15,000 compounds from the National Institutes of Health Molecular Libraries Screening Center Network was performed to validate its performance in a high-throughput screening format.
Subject(s)
Fluorescence Polarization/methods , HSP90 Heat-Shock Proteins/analysis , Neoplasms/chemistry , Cell Line, Tumor , HumansABSTRACT
Ethanolic extracts from fresh Echinacea purpurea and Spilanthes acmella and dried Hydrastis canadensis were examined with regard to their ability to inhibit cytochrome P450(2E1) mediated oxidation of p-nitrophenol in vitro. In addition, individual constituents of these extracts, including alkylamides from E. purpurea and S. acmella, caffeic acid derivatives from E. purpurea, and several of the major alkaloids from H. canadensis, were tested for inhibition using the same assay. H. canadensis (goldenseal) was a strong inhibitor of the P450(2E1), and the inhibition appeared to be related to the presence of the alkaloids berberine, hydrastine and canadine in the extract. These compounds inhibited 2E1 with K(I) values ranging from 2.8 microM for hydrastine to 18 microM for berberine. The alkylamides present in E. purpurea and S. acmella also showed significant inhibition at concentrations as low as 25 microM, whereas the caffeic acid derivatives had no effect. Commercial green tea preparations, along with four of the individual tea catechins, were also examined and were found to have no effect on the activity of P450(2E1).
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/drug effects , Phytotherapy , Plant Extracts/pharmacology , Plants, Medicinal , Baculoviridae/enzymology , Echinacea , Helianthus , Humans , Hydrastis , Microsomes, Liver/enzymology , Plant Extracts/administration & dosage , Plant RootsABSTRACT
Inactivation of NF2/Merlin causes the autosomal-dominant cancer predisposition syndrome familial neurofibromatosis type 2 (NF2) and contributes to the development of malignant pleural mesothelioma (MPM). To develop a targeted therapy for NF2-mutant tumors, we have exploited the recent realization that Merlin loss drives tumorigenesis by activating the E3 ubiquitin ligase CRL4DCAF1, thereby inhibiting the Hippo pathway component Lats. Here, we show that MLN4924, a NEDD8-activating enzyme (NAE) inhibitor, suppresses CRL4DCAF1 and attenuates activation of YAP in NF2-mutant tumor cells. In addition, MLN4924 sensitizes MPM to traditional chemotherapy, presumably as a result of collateral inhibition of cullin-RING ubiquitin ligases (CRL) involved in DNA repair. However, even in combination with chemotherapy, MLN4924 does not exhibit significant preclinical activity. Further analysis revealed that depletion of DCAF1 or treatment with MLN4924 does not affect mTOR hyperactivation in NF2-mutant tumor cells, suggesting that loss of Merlin activates mTOR independently of CRL4DCAF1 Intriguingly, combining MLN4924 with the mTOR/PI3K inhibitor GDC-0980 suppresses the growth of NF2-mutant tumor cells in vitro as well as in mouse and patient-derived xenografts. These results provide preclinical rationale for the use of NAE inhibitors in combination with mTOR/PI3K inhibitors in NF2-mutant tumors. Mol Cancer Ther; 16(8); 1693-704. ©2017 AACR.
Subject(s)
Carcinogenesis/metabolism , Carcinogenesis/pathology , Neurofibromin 2/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclopentanes/pharmacology , Cyclopentanes/therapeutic use , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mesothelioma/drug therapy , Mesothelioma/pathology , Mesothelioma, Malignant , Mice , Mutation/genetics , Oncogenes , Phosphatidylinositol 3-Kinases/metabolism , Pleural Neoplasms/drug therapy , Pleural Neoplasms/pathology , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Ubiquitin-Activating Enzymes/metabolismABSTRACT
Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.
Subject(s)
Breast Neoplasms/enzymology , Drug Design , HSP70 Heat-Shock Proteins/agonists , HSP70 Heat-Shock Proteins/metabolism , Immunoassay/methods , Neuroblastoma/enzymology , Neurons/enzymology , Cell Line, Tumor , Gene Expression Regulation, Enzymologic/drug effects , Humans , Neurons/drug effects , Up-Regulation/drug effectsABSTRACT
Neurodegeneration, a result of multiple dysregulatory events, is a lengthy multistep process manifested by accrual of mutant variants and abnormal expression, posttranslational modification, and processing of certain proteins. Accumulation of these dysregulated processes requires a mechanism that maintains their functional stability and allows the evolution of the neurodegenerative phenotype. In malignant cells, the capacity to buffer transformation has been attributed to heat-shock protein 90 (Hsp90). Although normal proteins seem to require limited assistance from the chaperone, their aberrant counterparts seem to be highly dependent on Hsp90. Whereas enhanced Hsp90 affinity for mutated or functionally deregulated client proteins has been observed for several oncoproteins, it is unknown whether Hsp90 plays a similar role for neuronal proteins and thus maintains and facilitates the transformed phenotype in neurodegenerative diseases. Tauopathies are neurodegenerative diseases characterized by aberrant phosphorylation and/or expression of Tau protein, leading to a time-dependent accumulation of Tau aggregates and subsequent neuronal death. Here, we show that the stability of p35, a neuronal protein that activates cyclin-dependent protein kinase 5 through complex formation leading to aberrant Tau phosphorylation, and that of mutant but not WT Tau protein is maintained in tauopathies by Hsp90. Inhibition of Hsp90 in cellular and mouse models of tauopathies leads to a reduction of the pathogenic activity of these proteins and results in elimination of aggregated Tau. The results identify important roles played by Hsp90 in maintaining and facilitating the degenerative phenotype in these diseases and provide a common principle governing cancer and neurodegenerative diseases.
Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Phenotype , Tauopathies/metabolism , Tauopathies/pathology , Animals , COS Cells , Chlorocebus aethiops , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , Mutation/genetics , Phosphorylation , Phosphotransferases/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Rats , Tauopathies/genetics , tau Proteins/genetics , tau Proteins/metabolismABSTRACT
The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.
Subject(s)
Apoptosis , Carcinoma, Small Cell/drug therapy , Carcinoma, Small Cell/metabolism , HSP90 Heat-Shock Proteins/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Antineoplastic Agents/pharmacology , Carcinoma, Small Cell/pathology , Cell Line, Tumor , Cell Transformation, Neoplastic , Dose-Response Relationship, Drug , Drug Design , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/pathology , Models, Chemical , Phosphatidylinositol 3-Kinases/metabolism , Time FactorsABSTRACT
The synthesis of a red-shifted cy3B-GM ligand and its evaluation as a fluorescence polarization probe for Hsp90 is presented.