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1.
Br J Cancer ; 110(10): 2479-88, 2014 May 13.
Article in English | MEDLINE | ID: mdl-24762959

ABSTRACT

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.


Subject(s)
Antineoplastic Agents/pharmacology , Mesothelioma/enzymology , Neoplasm Proteins/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , Butadienes/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Chromones/pharmacology , Drug Screening Assays, Antitumor , Enzyme Activation/drug effects , Everolimus , Humans , Imidazoles/pharmacology , Indazoles/pharmacology , MAP Kinase Signaling System , Mesothelioma/pathology , Molecular Targeted Therapy , Morpholines/pharmacology , Neoplasm Proteins/physiology , Nitriles/pharmacology , Phosphatidylinositol 3-Kinases/physiology , Quinolines/pharmacology , RNA Interference , RNA, Small Interfering/pharmacology , Receptor Protein-Tyrosine Kinases/physiology , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Sulfonamides/pharmacology , TOR Serine-Threonine Kinases/physiology , raf Kinases/physiology
2.
Oncogene ; 33(14): 1872-6, 2014 Apr 03.
Article in English | MEDLINE | ID: mdl-23584476

ABSTRACT

Most gastrointestinal stromal tumors (GISTs) contain KIT or PDGFRA kinase gain-of-function mutations, and therefore respond clinically to imatinib and other tyrosine kinase inhibitor (TKI) therapies. However, clinical progression subsequently results from selection of TKI-resistant clones, typically containing secondary mutations in the KIT kinase domain, which can be heterogeneous between and within GIST metastases in a given patient. TKI-resistant KIT oncoproteins require HSP90 chaperoning and are potently inactivated by HSP90 inhibitors, but clinical applications in GIST patients are constrained by the toxicity resulting from concomitant inactivation of various other HSP90 client proteins, beyond KIT and PDGFRA. To identify novel targets responsible for KIT oncoprotein function, we performed parallel genome-scale short hairpin RNA (shRNA)-mediated gene knockdowns in KIT-mutant GIST-T1 and GIST882. GIST cells were infected with a lentiviral shRNA pooled library targeting 11 194 human genes, and allowed to proliferate for 5-7 weeks, at which point assessment of relative hairpin abundance identified the HSP90 cofactor, CDC37, as one of the top six GIST-specific essential genes. Validations in treatment-naive (GIST-T1, GIST882) vs imatinib-resistant GISTs (GIST48, GIST430) demonstrated that: (1) CDC37 interacts with oncogenic KIT; (2) CDC37 regulates expression and activation of KIT and downstream signaling intermediates in GIST; and (3) unlike direct HSP90 inhibition, CDC37 knockdown accomplishes prolonged KIT inhibition (>20 days) in GIST. These studies highlight CDC37 as a key biologic vulnerability in both imatinib-sensitive and imatinib-resistant GIST. CDC37 targeting is expected to be selective for KIT/PDGFRA and a subset of other HSP90 clients, and thereby represents a promising strategy for inactivating the myriad KIT/PDGFRA oncoproteins in TKI-resistant GIST patients.


Subject(s)
Cell Cycle Proteins/metabolism , Chaperonins/metabolism , Gastrointestinal Stromal Tumors/metabolism , Gene Expression Regulation, Neoplastic , HSP90 Heat-Shock Proteins/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival , Drug Resistance, Neoplasm , Gene Expression Profiling , Gene Library , Humans , Lentivirus/metabolism , Oncogenes , Pentacyclic Triterpenes , Protein Kinase Inhibitors/chemistry , RNA, Small Interfering/metabolism , Triterpenes/chemistry
3.
Oncogene ; 30(14): 1643-52, 2011 Apr 07.
Article in English | MEDLINE | ID: mdl-21132014

ABSTRACT

Mesothelioma is an asbestos-associated and notoriously chemotherapy-resistant neoplasm. Activation of the receptor tyrosine kinases (RTKs), epidermal growth factor receptor and MET, has been described in subsets of mesothelioma, suggesting that TKs might represent therapeutic targets in this highly lethal disease. We employed proteomic screening by phosphotyrosine immunoaffinity purification and tandem mass spectrometry to characterize RTK activation in mesothelioma cell lines. These assays demonstrated expression and activation of the AXL protein, which is an RTK with known oncogenic properties in non-mesothelial cancer types. AXL was expressed and activated strongly in 8 of 9 mesothelioma cell lines and 6 of 12 mesothelioma biopsies, including each of 12 mesotheliomas with spindle-cell histology. Somatic AXL mutations were not found, but all mesotheliomas expressed an alternatively spliced AXL transcript with in-frame deletion of exon 10, and six of seven mesothelioma cell lines expressed the AXL ligand, growth arrest-specific 6 (GAS6). GAS6 expression appeared to be functionally relevant, as indicated by modulation of AXL tyrosine phosphorylation by knockdown of endogeneous GAS6, and by administration of exogenous GAS6. AXL silencing by lentivirus-mediated short hairpin RNA suppressed mesothelioma migration and cellular proliferation due to G1 arrest. The AXL inhibitor DP-3975 inhibited cell migration and proliferation in mesotheliomas with strong AXL activation. DP-3975 response in these tumors was characterized by inhibition of PI3-K/AKT/mTOR and RAF/MAPK signaling. AXL inhibition suppressed mesothelioma anchorage-independent growth, with reduction in colony numbers and size. These studies suggest that AXL inhibitors warrant clinical evaluation in mesothelioma.


Subject(s)
Cell Proliferation/drug effects , Mesothelioma/genetics , Pleural Neoplasms/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Alternative Splicing , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Exons , Gene Silencing , Humans , Intercellular Signaling Peptides and Proteins/isolation & purification , Mesothelioma/drug therapy , Mesothelioma/pathology , Neoplasm Invasiveness/genetics , Phosphorylation , Pleural Neoplasms/drug therapy , Pleural Neoplasms/pathology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Sequence Deletion , Signal Transduction/drug effects , Signal Transduction/genetics , Axl Receptor Tyrosine Kinase
4.
Oncogene ; 27(42): 5624-34, 2008 Sep 18.
Article in English | MEDLINE | ID: mdl-18521081

ABSTRACT

Oncogenic KIT or PDGFRA receptor tyrosine kinase mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is standard of care for patients with metastatic GIST. However, most of these patients eventually develop clinical resistance to imatinib and other KIT/PDGFRA kinase inhibitors and there is an urgent need to identify novel therapeutic strategies. We reported previously that protein kinase C-theta (PKCtheta) is activated in GIST, irrespective of KIT or PDGFRA mutational status, and is expressed at levels unprecedented in other mesenchymal tumors, therefore serving as a diagnostic marker of GIST. Herein, we characterize biological functions of PKCtheta in imatinib-sensitive and imatinib-resistant GISTs, showing that lentivirus-mediated PKCtheta knockdown is accompanied by inhibition of KIT expression in three KIT+/PKCtheta+ GIST cell lines, but not in a comparator KIT+/PKCtheta- Ewing's sarcoma cell line. PKCtheta knockdown in the KIT+ GISTs was associated with inhibition of the phosphatidylinositol-3-kinase/AKT signaling pathway, upregulation of the cyclin-dependent kinase inhibitors p21 and p27, antiproliferative effects due to G(1) arrest and induction of apoptosis, comparable to the effects seen after direct knockdown of KIT expression by KIT short-hairpin RNA. These novel findings highlight that PKCtheta warrants clinical evaluation as a potential therapeutic target in GISTs, including those cases containing mutations that confer resistance to KIT/PDGFRA kinase inhibitors.


Subject(s)
Gastrointestinal Stromal Tumors/pathology , Isoenzymes/physiology , Protein Kinase C/physiology , Proto-Oncogene Proteins c-kit/physiology , Apoptosis , Base Sequence , Benzamides , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Humans , Imatinib Mesylate , Isoenzymes/genetics , Molecular Sequence Data , Phosphorylation , Piperazines/pharmacology , Protein Kinase C/genetics , Protein Kinase C-theta , Proto-Oncogene Proteins c-kit/genetics , Pyrimidines/pharmacology , RNA, Small Interfering/genetics , Signal Transduction
5.
Oncogene ; 26(44): 6386-95, 2007 Sep 27.
Article in English | MEDLINE | ID: mdl-17452978

ABSTRACT

Most gastrointestinal stromal tumors (GISTs) express oncogenic and constitutively active forms of the KIT or platelet-derived growth factor receptor alpha (PDGFRA) receptor tyrosine kinase proteins, and these kinase oncoproteins serve as targets for effective therapies. Given that mutant KIT oncoproteins serve crucial transforming roles in GISTs, we evaluated interactions with the KIT oncoproteins and determined signaling pathways that are dependent on KIT oncogenic activation in GISTs. Tyrosine-phosphorylated KIT oncoproteins interacted with PDGFRA, PDGFRB, phosphatidylinositol 3-kinase (PI3-K) and PKCtheta in GIST cells, and these interactions were abolished by KIT inhibition with imatinib or PKC412 or KIT RNAi. Notably, tyrosine-phosphorylated PDGFRA was prominent in frozen GIST tumors expressing KIT oncoproteins, suggesting that KIT-mediated PDGFRA phosphorylation is an efficient and biologically consequential mechanism in GISTs. Activated signaling intermediates were identified by immunoaffinity purification of tyrosine-phosphorylated proteins in GIST cells before and after treatment with KIT inhibitors, and these analyses show that GRB2, SHC, CBL and MAPK activation are largely KIT dependent in GISTs, whereas PI3-K, STAT1 and STAT3 activation are partially KIT dependent. In addition, we found that phosphorylation of several tyrosine kinase proteins - including JAK1 and EPHA4 - did not depend on KIT activation. Likewise, paxillin activation was independent of the KIT oncogenic signal. These studies identify signaling pathways that can provide both KIT-dependent and KIT-independent therapeutic synergies in GIST, and thereby highlight clinical strategies that might consolidate GIST therapeutic response to KIT/PDGFRA inhibition.


Subject(s)
Gastrointestinal Stromal Tumors/metabolism , Protein Kinase C-delta/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Antineoplastic Agents/pharmacology , Blotting, Western , Drug Resistance, Neoplasm , Gastrointestinal Stromal Tumors/genetics , Gastrointestinal Stromal Tumors/pathology , Humans , Immunoprecipitation , Mutation , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Kinase C-delta/genetics , Proto-Oncogene Proteins c-kit/drug effects , Proto-Oncogene Proteins c-kit/genetics , RNA, Small Interfering/pharmacology , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptor, Platelet-Derived Growth Factor beta/genetics , Receptor, Platelet-Derived Growth Factor beta/metabolism , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Tumor Cells, Cultured , Tyrosine/metabolism
6.
Biochemistry (Mosc) ; 67(5): 547-52, 2002 May.
Article in English | MEDLINE | ID: mdl-12059774

ABSTRACT

Chaperone GroEL was treated with different concentrations of hydrogen peroxide. The conformational states of GroEL were monitored by protein intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate fluorescence, and far-UV CD measurements. The results show that GroEL has unusual ability to resist oxidative stress. GroEL kept its quaternary structure and activity even when treated with 10 mM hydrogen peroxide. Two fragments were formed when GroEL was treated with high concentrations of hydrogen peroxide (more than 20 mM). It is suggested that GroEL, as a molecular chaperone, is related to oxidative process in vivo.


Subject(s)
Chaperonin 60/chemistry , Chaperonin 60/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli , Hydrogen Peroxide/pharmacology , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Oxidative Stress , Protein Conformation/drug effects , Solutions , Spectrometry, Fluorescence
7.
Eur J Biochem ; 268(22): 5901-11, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11722578

ABSTRACT

The effects of osmolytes, including dimethysulfoxide, sucrose, glycine and proline, on the unfolding and inactivation of guanidine-denatured creatine kinase were studied by observing the fluorescence emission spectra, the CD spectra and the inactivation of enzymatic activity. The results showed that low concentrations of dimethysulfoxide (< 40%), glycine (< 1.5 m), proline (< 2.5 m) and sucrose (< 1.2 m) reduced the inactivation and unfolding rate constants of creatine kinase, increased the change in transition free energy of inactivation and unfolding (Delta Delta G(u)) and stabilized its active conformation relative to the partially unfolded state with no osmolytes. In the presence of various osmolytes, the inactivation and unfolding dynamics of creatine kinase were related to the protein concentrations. These osmolytes protected creatine kinase against guanidine denaturation in a concentration-dependent manner. The ability of the osmolytes to protect creatine kinase against guanidine denaturation decreased in order from sucrose to glycine to proline. Dimethysulfoxide was considered separately. This study also suggests that osmolytes are not only energy substrates for metabolism and organic components in vivo, but also have an important physiological function for maintaining adequate rates of enzymatic catalysis and for stabilizing the protein secondary and tertiary conformations.


Subject(s)
Creatine Kinase/chemistry , Guanidine/chemistry , Anilino Naphthalenesulfonates/chemistry , Animals , Creatine Kinase/antagonists & inhibitors , Fluorescent Dyes/chemistry , Muscles/enzymology , Osmolar Concentration , Protein Denaturation , Protein Structure, Secondary , Rabbits , Spectrometry, Fluorescence , Sucrose/chemistry
8.
Protein Sci ; 10(11): 2346-53, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11604540

ABSTRACT

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.


Subject(s)
Creatine Kinase/chemistry , Cyclophilin A/chemistry , Molecular Chaperones/chemistry , Dimerization , Drug Storage , Enzyme Activation , Hydrogen-Ion Concentration , Protein Folding , Spectrometry, Fluorescence , Temperature
9.
Biochem Cell Biol ; 79(4): 479-87, 2001.
Article in English | MEDLINE | ID: mdl-11527217

ABSTRACT

Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.


Subject(s)
Creatine Kinase/metabolism , Muscles/enzymology , Anilino Naphthalenesulfonates , Animals , Chaperonin 60/chemistry , Chaperonin 60/metabolism , Circular Dichroism , Creatine Kinase/chemistry , Dinitrobenzenes , Electrophoresis, Polyacrylamide Gel , Guanidine , Kinetics , Protein Denaturation , Protein Folding , Rabbits , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet
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