ABSTRACT
Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.
Subject(s)
Neoplasms/genetics , Neoplasms/pathology , RNA Interference , Cell Line, Tumor , Gene Library , Gene Regulatory Networks , Humans , Multiprotein Complexes/metabolism , Neoplasms/metabolism , Oncogenes , RNA, Small Interfering , Signal Transduction , Transcription Factors/metabolismABSTRACT
Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1-mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in â¼10-15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of "cancer-selective paralog dependency" may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.
Subject(s)
DNA Helicases/deficiency , Epigenesis, Genetic/physiology , Multiprotein Complexes/genetics , Neoplasms/genetics , Nuclear Proteins/deficiency , Transcription Factors/deficiency , Transcription Factors/genetics , Blotting, Western , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cellular Senescence/genetics , Gene Knockdown Techniques , Gene Library , Histones/metabolism , Humans , Immunoprecipitation , Multiprotein Complexes/metabolism , RNA, Small Interfering/genetics , Transcription Factors/metabolismABSTRACT
High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns.
Subject(s)
RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transfection , Cell Line , Cell Line, Tumor , HeLa Cells , HumansABSTRACT
By combining the use of BD Biosciences FluoroBlok membrane-based Boyden chambers with the Cellomics HCS ArrayScan, a more sensitive method for measuring cell migration has been developed. This assay is based on counting nuclei of migrated cells on the bottom of the filter rather than conventional approaches, which use measurement of total well fluorescence. This cell migration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor-mediated endothelial cell migration.
Subject(s)
Cell Movement , Endothelial Cells/physiology , High-Throughput Screening Assays/methods , Cell Movement/drug effects , Human Umbilical Vein Endothelial Cells/physiology , Humans , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers a variety of proinflammatory responses. However, our previous work revealed that RAGE-binding AGEs free of endotoxin were incapable of inducing vascular cell adhesion molecule-1 (VCAM-1) or tumor necrosis factor-alpha (TNF-alpha) expression. Thus, the objective of this study was to clarify the role of AGEs in cell activation through gene expression profiling using both in vitro and in vivo model systems. Endothelial cells treated with AGE-BSA, previously shown to bind RAGE with high affinity, did not show gene expression changes indicative of an inflammatory response. In contrast, the alternate RAGE ligand, S100b, triggered an increase in endothelial mRNA expression of a variety of immune-related genes. The effects of AGEs were studied in vivo using healthy mice exposed to two different treatment conditions: 1) intravenous injection of a single dose of model AGEs or 2) four intraperitoneal injections of model AGEs (once per day). In both cases, the liver was extracted for gene expression profiling. Both of the short-term AGE treatments resulted in a moderate increase in liver mRNA levels for genes involved in macrophage-based clearance/detoxification of foreign agents. Our findings using AGEs with strong RAGE-binding properties indicate that AGEs may not uniformly play a role in cellular activation.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation/physiology , Glycation End Products, Advanced/metabolism , Nerve Growth Factors/genetics , S100 Proteins/genetics , Animals , Autoantigens/genetics , Cattle , Enzymes/genetics , Liver/physiology , Mice , Proteins/genetics , RNA, Messenger/genetics , Receptor for Advanced Glycation End Products , Receptors, Immunologic , S100 Calcium Binding Protein beta Subunit , Serum Albumin, Bovine/metabolism , Tumor Necrosis Factor-alpha/genetics , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
By combining the use of BD Biosciences FluoroBlok membrane-based Boyden chambers with the Cellomics HCS ArrayScan, a more sensitive method for measuring cell migration has been developed. This assay is based on counting nuclei of migrated cells on the bottom of the filter rather than conventional approaches, which use measurement of total well fluorescence. This cell migration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor-mediated endothelial cell migration.
Subject(s)
Angiogenesis Inducing Agents/pharmacology , Angiogenesis Inhibitors/pharmacology , Cell Movement/drug effects , Drug Evaluation, Preclinical/methods , Endothelium, Vascular/drug effects , Vascular Endothelial Growth Factors/pharmacology , Biological Assay , Cell Movement/physiology , Cell Nucleus/physiology , Endothelium, Vascular/chemistry , Endothelium, Vascular/physiology , Fluoresceins/chemistry , Humans , Umbilical Cord/cytology , Vascular Endothelial Growth Factors/physiologyABSTRACT
In the current study, we isolated sublines of the human breast adenocarcinoma cell line MDA 435 that exhibited increasing resistance to epothilone A, a microtubule-stabilizing cytotoxic agent. The resistant cells did not express P glycoprotein or multidrug resistance-associated protein (MRP) which are known mediators of multidrug resistance (MDR). Two groups of epothilone A-resistant cells were selected: cells which exhibited low resistance to both epothilone A and Taxol, and cells which exhibit low resistance to Taxol but high resistance to epothilone A. cDNA microarrays of epothilone A-resistant and Taxol-resistant cells were utilized to further characterize epothilone A resistance. Hierarchical clustering of genes according to their levels of expression indicated that the majority of genes which were highly expressed in epothilone A-resistant cells but not in taxol-resistant MDR cells encode known interferon-inducible proteins. Genes whose expression increased with increasing epothilone A resistance include microtubule-associated GTPases, cytoskeletal proteins, cell signalling proteins and a drug metabolising enzyme. The majority of the genes that were repressed in both epothilone A- and Taxol-resistant cells encode proteins regulating cellular growth signalling mechanisms.
Subject(s)
Antineoplastic Agents/pharmacology , Epothilones/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/genetics , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cytoskeletal Proteins/biosynthesis , Cytoskeletal Proteins/genetics , Drug Resistance, Neoplasm/genetics , Female , GTP Phosphohydrolases/biosynthesis , GTP Phosphohydrolases/genetics , Gene Expression Regulation, Neoplastic/drug effects , Growth Substances/biosynthesis , Growth Substances/genetics , HL-60 Cells/drug effects , Humans , Interferons/pharmacology , Male , Microtubules/drug effects , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/physiology , Paclitaxel/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolismABSTRACT
Using a gene expression analysis approach we found that the mRNA encoding the lysosomal cysteine protease cathepsin S (CatS) was up-regulated in rat dorsal root ganglia (DRG) following peripheral nerve injury. CatS protein was expressed in infiltrating macrophages in DRG and near the site of injury. At both sites CatS expression progressively increased from day 3 to day 14 after injury. In naïve rats, intraplantar injection of activated rat recombinant (rr) CatS (0.3, 1 microg/rat) induced a mechanical hyperalgesia that developed within half-an-hour, diminished by 3h and was absent after 24h. Activated rrCathepsin B (CatB) and non-activated rrCatS injected intraplantarly at the same or higher doses than activated rrCatS had no effect on rat nociceptive thresholds. In nerve-injured rats, mechanical hyperalgesia, but not allodynia, was significantly reversed for up to 3h by systemic administration of a non-brain penetrant, irreversible CatS inhibitor (LHVS, 3-30 mg/kg s.c.). Depletion of peripheral macrophages by intravenous injection of liposome encapsulate clodronate (1ml, 5 mg/ml) partially reduced established mechanical hyperalgesia but not allodynia, and abolished the anti-hyperalgesic effect of LHVS. Our results demonstrate a pro-nociceptive effect of CatS and indicate that endogenous CatS released by peripheral macrophages contributes to the maintenance of neuropathic hyperalgesia following nerve injury.
Subject(s)
Cathepsins/genetics , Cathepsins/metabolism , Hyperalgesia/metabolism , Hyperalgesia/physiopathology , Sciatic Nerve/enzymology , Animals , Cathepsins/pharmacology , Chronic Disease , Cysteine Endopeptidases/metabolism , Disease Models, Animal , Gene Expression Regulation, Enzymologic , Hyperalgesia/immunology , Ligation , Macrophages/enzymology , Male , Nociceptors/drug effects , Nociceptors/enzymology , Nociceptors/immunology , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Rats , Rats, Wistar , Sciatic Nerve/immunology , Sciatic Nerve/physiopathology , Sciatica/immunology , Sciatica/metabolism , Sciatica/physiopathologyABSTRACT
This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.