RESUMO
Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine CD8+ T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.
Assuntos
Sistemas CRISPR-Cas , Neoplasias , Humanos , Animais , Camundongos , RNA Guia de Sistemas CRISPR-Cas , Linfócitos do Interstício Tumoral , Imunoterapia Adotiva , Neoplasias/genética , Edição de Genes , Proteína 1 Supressora da Sinalização de Citocina/genéticaRESUMO
Interferons (IFNs) are cytokines that play a critical role in limiting infectious and malignant diseases 1-4 . Emerging data suggest that the strength and duration of IFN signaling can differentially impact cancer therapies, including immune checkpoint blockade 5-7 . Here, we characterize the output of IFN signaling, specifically IFN-stimulated gene (ISG) signatures, in primary tumors from The Cancer Genome Atlas. While immune infiltration correlates with the ISG signature in some primary tumors, the existence of ISG signature-positive tumors without evident infiltration of IFN-producing immune cells suggests that cancer cells per se can be a source of IFN production. Consistent with this hypothesis, analysis of patient-derived tumor xenografts propagated in immune-deficient mice shows evidence of ISG-positive tumors that correlates with expression of human type I and III IFNs derived from the cancer cells. Mechanistic studies using cell line models from the Cancer Cell Line Encyclopedia that harbor ISG signatures demonstrate that this is a by-product of a STING-dependent pathway resulting in chronic tumor-derived IFN production. This imposes a transcriptional state on the tumor, poising it to respond to the aberrant accumulation of double-stranded RNA (dsRNA) due to increased sensor levels (MDA5, RIG-I and PKR). By interrogating our functional short-hairpin RNA screen dataset across 398 cancer cell lines, we show that this ISG transcriptional state creates a novel genetic vulnerability. ISG signature-positive cancer cells are sensitive to the loss of ADAR, a dsRNA-editing enzyme that is also an ISG. A genome-wide CRISPR genetic suppressor screen reveals that the entire type I IFN pathway and the dsRNA-activated kinase, PKR, are required for the lethality induced by ADAR depletion. Therefore, tumor-derived IFN resulting in chronic signaling creates a cellular state primed to respond to dsRNA accumulation, rendering ISG-positive tumors susceptible to ADAR loss.
Assuntos
Adenosina Desaminase/metabolismo , Interferons/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos Nus , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Supressão Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Perturbed epigenomic programs play key roles in tumorigenesis, and chromatin modulators are candidate therapeutic targets in various human cancer types. To define singular and shared dependencies on DNA and histone modifiers and transcription factors in poorly differentiated adult and pediatric cancers, we conducted a targeted shRNA screen across 59 cell lines of 6 cancer types. Here, we describe the TRPS1 transcription factor as a strong breast cancer-specific hit, owing largely to lineage-restricted expression. Knockdown of TRPS1 resulted in perturbed mitosis, apoptosis, and reduced tumor growth. Integrated analysis of TRPS1 transcriptional targets, chromatin binding, and protein interactions revealed that TRPS1 is associated with the NuRD repressor complex. These findings uncover a transcriptional network that is essential for breast cancer cell survival and propagation.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Feminino , Células HEK293 , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Senescence is a terminal differentiation program that halts the growth of damaged cells and must be circumvented for cancer to arise. Here we describe a panel of genetic screens to identify genes required for replicative senescence. We uncover a role in senescence for the potent tumor suppressor and ATM substrate USP28. USP28 controls activation of both the TP53 branch and the GATA4/NFkB branch that controls the senescence-associated secretory phenotype (SASP). These results suggest a role for ubiquitination in senescence and imply a common node downstream from ATM that links the TP53 and GATA4 branches of the senescence response.
Assuntos
Senescência Celular/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Fator de Transcrição GATA4/genética , Biblioteca Gênica , Células HCT116 , Humanos , Reprodutibilidade dos Testes , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genética , UbiquitinaçãoRESUMO
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.
Assuntos
Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Linhagem Celular Tumoral , Biblioteca Gênica , Redes Reguladoras de Genes , Humanos , Complexos Multiproteicos/metabolismo , Neoplasias/metabolismo , Oncogenes , RNA Interferente Pequeno , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs (Balak et al., 2006; Kosaka et al., 2006; Rudin et al., 2013; Wagle et al., 2011). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of KEAP1, a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in the presence of multiple inhibitors targeting the RTK/Ras/MAPK pathway.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas de Inativação de Genes , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
UNLABELLED: CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. We compared the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies across multiple cancer cell lines. CRISPR dropout screens consistently identified more lethal genes than RNAi, implying that the identification of many cellular dependencies may require full gene inactivation. However, in two aneuploid cancer models, we found that all genes within highly amplified regions, including nonexpressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits. In addition, using a CRISPR tiling screen, we found that sgRNAs targeting essential domains generate the strongest lethality phenotypes and thus provide a strategy to rapidly define the protein domains required for cancer dependence. Collectively, these findings not only demonstrate the utility of CRISPR screens in the identification of cancer-essential genes, but also reveal the need to carefully control for false-positive results in chromosomally unstable cancer lines. SIGNIFICANCE: We show in this study that CRISPR-based screens have a significantly lower false-negative rate compared with RNAi-based screens, but have specific liabilities particularly in the interrogation of regions of genome amplification. Therefore, this study provides critical insights for applying CRISPR-based screens toward the systematic identification of new cancer targets. Cancer Discov; 6(8); 900-13. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Aguirre et al., p. 914This article is highlighted in the In This Issue feature, p. 803.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Amplificação de Genes , Genoma Humano , Genômica , Neoplasias/genética , Linhagem Celular Tumoral , Estudos de Associação Genética , Genômica/métodos , Genômica/normas , Ensaios de Triagem em Larga Escala , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , RNA Guia de Cinetoplastídeos/genética , RNA Interferente Pequeno/genética , Reprodutibilidade dos TestesRESUMO
5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metionina/metabolismo , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Desoxiadenosinas/metabolismo , Deleção de Genes , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína-Arginina N-Metiltransferases/genética , Purina-Núcleosídeo Fosforilase/genética , RNA Interferente Pequeno/genética , Tionucleosídeos/metabolismoRESUMO
Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Diferenciação Celular , Linhagem Celular Tumoral , Crizotinibe , DNA/química , DNA Complementar/metabolismo , Transição Epitelial-Mesenquimal , Cloridrato de Erlotinib , Proteínas de Fusão bcr-abl/genética , Dosagem de Genes , Biblioteca Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Modelos Teóricos , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Quinazolinas/administração & dosagem , Análise de Sequência de RNARESUMO
Assessing the functional significance of novel putative oncogenes remains a significant challenge given the limitations of current loss-of-function tools. Here, we describe a method that employs TALEN or CRISPR/Cas9-mediated knock-in of inducible degron tags (Degron-KI) that provides a versatile approach for the functional characterization of novel cancer genes and addresses many of the shortcomings of current tools. The Degron-KI system allows for highly specific, inducible, and allele-targeted inhibition of endogenous protein function, and the ability to titrate protein depletion with this system is able to better mimic pharmacologic inhibition compared with RNAi or genetic knockout approaches. The Degron-KI system was able to faithfully recapitulate the effects of pharmacologic EZH2 and PI3Kα inhibitors in cancer cell lines. The application of this system to the study of a poorly understood putative oncogene, SF3B1, provided the first causal link between SF3B1 hotspot mutations and splicing alterations. Surprisingly, we found that SF3B1-mutant cells are not dependent upon the mutated allele for in vitro growth, but instead depend upon the function of the remaining wild-type alleles. Collectively, these results demonstrate the broad utility of the Degron-KI system for the functional characterization of cancer genes.
Assuntos
Genes Neoplásicos , Neoplasias/genética , Proliferação de Células , Células HCT116 , Humanos , Mutação , Fosfoproteínas/genética , Estabilidade Proteica , Proteólise , Fatores de Processamento de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genéticaRESUMO
Glioblastoma remains one of the deadliest of human cancers, with most patients succumbing to the disease within two years of diagnosis. The available data suggest that simultaneous inactivation of critical nodes within the glioblastoma molecular circuitry will be required for meaningful clinical efficacy. We conducted parallel genome-wide shRNA screens to identify such nodes and uncovered a number of G-Protein Coupled Receptor (GPCR) neurotransmitter pathways, including the Dopamine Receptor D2 (DRD2) signaling pathway. Supporting the importance of DRD2 in glioblastoma, DRD2 mRNA and protein expression were elevated in clinical glioblastoma specimens relative to matched non-neoplastic cerebrum. Treatment with independent si-/shRNAs against DRD2 or with DRD2 antagonists suppressed the growth of patient-derived glioblastoma lines both in vitro and in vivo. Importantly, glioblastoma lines derived from independent genetically engineered mouse models (GEMMs) were more sensitive to haloperidol, an FDA approved DRD2 antagonist, than the premalignant astrocyte lines by approximately an order of magnitude. The pro-proliferative effect of DRD2 was, in part, mediated through a GNAI2/Rap1/Ras/ERK signaling axis. Combined inhibition of DRD2 and Epidermal Growth Factor Receptor (EGFR) led to synergistic tumoricidal activity as well as ERK suppression in independent in vivo and in vitro glioblastoma models. Our results suggest combined EGFR and DRD2 inhibition as a promising strategy for glioblastoma treatment.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , RNA Interferente Pequeno/genética , Receptores de Dopamina D2/metabolismo , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/patologia , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Fosforilação , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Tumors exhibit numerous recurrent hemizygous focal deletions that contain no known tumor suppressors and are poorly understood. To investigate whether these regions contribute to tumorigenesis, we searched genetically for genes with cancer-relevant properties within these hemizygous deletions. We identified STOP and GO genes, which negatively and positively regulate proliferation, respectively. STOP genes include many known tumor suppressors, whereas GO genes are enriched for essential genes. Analysis of their chromosomal distribution revealed that recurring deletions preferentially overrepresent STOP genes and underrepresent GO genes. We propose a hypothesis called the cancer gene island model, whereby gene islands encompassing high densities of STOP genes and low densities of GO genes are hemizygously deleted to maximize proliferative fitness through cumulative haploinsufficiencies. Because hundreds to thousands of genes are hemizygously deleted per tumor, this mechanism may help to drive tumorigenesis across many cancer types.
Assuntos
Proliferação de Células , Transformação Celular Neoplásica , Genes Neoplásicos , Haploinsuficiência , Neoplasias/genética , Neoplasias/patologia , Deleção de Sequência , Linhagem Celular , Linhagem Celular Tumoral , Mapeamento Cromossômico , Genes Essenciais , Genes Recessivos , Genes Supressores de Tumor , Hemizigoto , Humanos , Modelos Genéticos , OncogenesRESUMO
Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search for Myc-synthetic lethal genes and uncovered a role for the SUMO-activating enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.
Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica , Genes myc , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Enzimas Ativadoras de Ubiquitina/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Ciclo Celular , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/mortalidade , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Mitose , Transplante de Neoplasias , Interferência de RNA , RNA Interferente Pequeno , Fuso Acromático/fisiologia , Sumoilação , Transplante Heterólogo , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible ß-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/ß-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon ß-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/ß-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear ß-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/ß-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/ß-catenin pathway.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Genes APC , Mutação , Via de Sinalização Wnt , beta Catenina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Ciclo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Interferente Pequeno/genética , Transdução de Sinais , Transplante Heterólogo , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
Replication stress involving collision of replisomes with camptothecin (CPT)-stabilized DNA-Topoisomerase I adducts activates an ATR-dependent pathway to promote repair by homologous recombination. To identify human genes that protect cells from such replication stress, we performed a genome-wide CPT sensitivity screen. Among numerous candidate genes are two previously unstudied proteins: the ankyrin repeat protein NFKBIL2 and C6ORF167 (MMS22L), distantly related to yeast replication stress regulator Mms22p. MMS22L and NFKBIL2 interact with each other and with FACT (facilitator of chromatin transcription) and MCM (minichromosome maintenance) complexes. Cells depleted of NFKBIL2 or MMS22L are sensitive to DNA-damaging agents, load phosphorylated RPA onto chromatin in a CTIP-dependent manner, activate the ATR/ATRIP-CHK1 and double-strand break repair signaling pathways, and are defective in HR. This study identifies MMS22L-NFKBIL2 as components of the replication stress control pathway and provides a resource for discovery of additional components of this pathway.
Assuntos
Camptotecina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Testes Genéticos , Genoma Humano/genética , Instabilidade Genômica/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células HeLa , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multienzimáticos/metabolismo , NF-kappa B/deficiência , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Proteína de Replicação A/metabolismo , Reprodutibilidade dos Testes , Estresse Fisiológico/efeitos dos fármacos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Oncogenic mutations in the small GTPase Ras are highly prevalent in cancer, but an understanding of the vulnerabilities of these cancers is lacking. We undertook a genome-wide RNAi screen to identify synthetic lethal interactions with the KRAS oncogene. We discovered a diverse set of proteins whose depletion selectively impaired the viability of Ras mutant cells. Among these we observed a strong enrichment for genes with mitotic functions. We describe a pathway involving the mitotic kinase PLK1, the anaphase-promoting complex/cyclosome, and the proteasome that, when inhibited, results in prometaphase accumulation and the subsequent death of Ras mutant cells. Gene expression analysis indicates that reduced expression of genes in this pathway correlates with increased survival of patients bearing tumors with a Ras transcriptional signature. Our results suggest a previously underappreciated role for Ras in mitotic progression and demonstrate a pharmacologically tractable pathway for the potential treatment of cancers harboring Ras mutations.
Assuntos
Neoplasias do Colo/metabolismo , Mitose , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Genoma Humano , Humanos , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Inibidores de Proteassoma , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras) , Interferência de RNA , Transdução de Sinais , Transplante Heterólogo , Quinase 1 Polo-LikeRESUMO
By virtue of their accumulated genetic alterations, tumor cells may acquire vulnerabilities that create opportunities for therapeutic intervention. We have devised a massively parallel strategy for screening short hairpin RNA (shRNA) collections for stable loss-of-function phenotypes. We assayed from 6000 to 20,000 shRNAs simultaneously to identify genes important for the proliferation and survival of five cell lines derived from human mammary tissue. Lethal shRNAs common to these cell lines targeted many known cell-cycle regulatory networks. Cell line-specific sensitivities to suppression of protein complexes and biological pathways also emerged, and these could be validated by RNA interference (RNAi) and pharmacologically. These studies establish a practical platform for genome-scale screening of complex phenotypes in mammalian cells and demonstrate that RNAi can be used to expose genotype-specific sensitivities.
Assuntos
Neoplasias da Mama/genética , Mama/citologia , Mama/metabolismo , Proliferação de Células , Sobrevivência Celular , Genes Essenciais , Redes e Vias Metabólicas/genética , Interferência de RNA , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Genômica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Interferente PequenoRESUMO
Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.
Assuntos
Neoplasias da Mama/genética , Proliferação de Células , Neoplasias do Colo/genética , Genes Neoplásicos , Genômica/métodos , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Neoplasias do Colo/patologia , Biblioteca Gênica , Vetores Genéticos , Genoma Humano , Humanos , MicroRNAs , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno , Retroviridae/genéticaRESUMO
Tumorigenesis is a multistep process characterized by a myriad of genetic and epigenetic alterations. Identifying the causal perturbations that confer malignant transformation is a central goal in cancer biology. Here we report an RNAi-based genetic screen for genes that suppress transformation of human mammary epithelial cells. We identified genes previously implicated in proliferative control and epithelial cell function including two established tumor suppressors, TGFBR2 and PTEN. In addition, we uncovered a previously unrecognized tumor suppressor role for REST/NRSF, a transcriptional repressor of neuronal gene expression. Array-CGH analysis identified REST as a frequent target of deletion in colorectal cancer. Furthermore, we detect a frameshift mutation of the REST gene in colorectal cancer cells that encodes a dominantly acting truncation capable of transforming epithelial cells. Cells lacking REST exhibit increased PI(3)K signaling and are dependent upon this pathway for their transformed phenotype. These results implicate REST as a human tumor suppressor and provide a novel approach to identifying candidate genes that suppress the development of human cancer.