RESUMO
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
We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.
Assuntos
Perfilação da Expressão Gênica/métodos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica/economia , Humanos , Neoplasias/tratamento farmacológico , Especificidade de Órgãos , Preparações Farmacêuticas/metabolismo , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/métodos , Bibliotecas de Moléculas PequenasRESUMO
The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.
Assuntos
Azepinas/farmacologia , Descoberta de Drogas , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Megacariócitos/metabolismo , Poliploidia , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Aurora Quinase A , Aurora Quinases , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Megacarioblástica Aguda/genética , Megacariócitos/citologia , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.
Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/metabolismo , Proteínas Virais/metabolismo , Apoptose , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Interferons/metabolismo , Pulmão/citologia , Pulmão/virologia , Proteômica , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/metabolismo , Proteínas Wnt/metabolismoRESUMO
An alternative to therapeutic targeting of oncogenes is to perform "synthetic lethality" screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of mutant KRAS-driven cancers and demonstrate the potential of RNAi screens for discovering functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers with "undruggable" genetic alterations.
Assuntos
Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Camundongos , Mutação , Células NIH 3T3 , Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras) , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkappaB kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappaB signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer.
Assuntos
Genes ras/genética , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Alelos , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Perfilação da Expressão Gênica , Genes Letais , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-rel/metabolismo , Transdução de Sinais , Proteína bcl-X/metabolismoRESUMO
Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
Assuntos
Clonagem Molecular/métodos , Vetores Genéticos/genética , Biblioteca Genômica , Lentivirus/genética , Humanos , Fases de Leitura AbertaRESUMO
Aberrant activation of the canonical WNT/beta-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Although dysregulated beta-catenin activity drives colon tumorigenesis, further genetic perturbations are required to elaborate full malignant transformation. To identify genes that both modulate beta-catenin activity and are essential for colon cancer cell proliferation, we conducted two loss-of-function screens in human colon cancer cells and compared genes identified in these screens with an analysis of copy number alterations in colon cancer specimens. One of these genes, CDK8, which encodes a member of the mediator complex, is located at 13q12.13, a region of recurrent copy number gain in a substantial fraction of colon cancers. Here we show that the suppression of CDK8 expression inhibits proliferation in colon cancer cells characterized by high levels of CDK8 and beta-catenin hyperactivity. CDK8 kinase activity was necessary for beta-catenin-driven transformation and for expression of several beta-catenin transcriptional targets. Together these observations suggest that therapeutic interventions targeting CDK8 may confer a clinical benefit in beta-catenin-driven malignancies.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Regulação Neoplásica da Expressão Gênica , Oncogenes , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Neoplasias Colorretais/patologia , Quinase 8 Dependente de Ciclina , Quinases Ciclina-Dependentes/deficiência , Dosagem de Genes , Humanos , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Interferência de RNA , Transcrição GênicaRESUMO
Many biological pathways were first uncovered by identifying mutants with visible phenotypes and by scoring every sample in a screen via tedious and subjective visual inspection. Now, automated image analysis can effectively score many phenotypes. In practical application, customizing an image-analysis algorithm or finding a sufficient number of example cells to train a machine learning algorithm can be infeasible, particularly when positive control samples are not available and the phenotype of interest is rare. Here we present a supervised machine learning approach that uses iterative feedback to readily score multiple subtle and complex morphological phenotypes in high-throughput, image-based screens. First, automated cytological profiling extracts hundreds of numerical descriptors for every cell in every image. Next, the researcher generates a rule (i.e., classifier) to recognize cells with a phenotype of interest during a short, interactive training session using iterative feedback. Finally, all of the cells in the experiment are automatically classified and each sample is scored based on the presence of cells displaying the phenotype. By using this approach, we successfully scored images in RNA interference screens in 2 organisms for the prevalence of 15 diverse cellular morphologies, some of which were previously intractable.
Assuntos
Algoritmos , Inteligência Artificial , Células , Citometria por Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Animais , Células/química , Células/citologia , Células/ultraestrutura , Diagnóstico por Imagem/métodos , Retroalimentação , Humanos , Reconhecimento Automatizado de Padrão/métodos , Fenótipo , Interferência de RNA , Análise Serial de TecidosRESUMO
An imidazolone â triazolone replacement addressed the limited passive permeability of a series of protein arginine methyl transferase 5 (PRMT5) inhibitors. This increase in passive permeability was unexpected given the increase in the hydrogen bond acceptor (HBA) count and topological polar surface area (TPSA), two descriptors that are typically inversely correlated with permeability. Quantum mechanics (QM) calculations revealed that this unusual effect was due to an electronically driven disconnect between TPSA and 3D-PSA, which manifests in a reduction in overall HBA strength as indicated by the HBA moment descriptor from COSMO-RS (conductor-like screening model for real solvation). HBA moment was subsequently deployed as a design parameter leading to the discovery of inhibitors with not only improved passive permeability but also reduced P-glycoprotein (P-gp) transport. Our case study suggests that hidden polarity as quantified by TPSA-3DPSA can be rationally designed through QM calculations.
Assuntos
Arginina , Antígeno Prostático Específico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Humanos , Masculino , Permeabilidade , Antígeno Prostático Específico/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Transferases/metabolismoRESUMO
SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.
Assuntos
Imunidade Celular , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais/genéticaRESUMO
Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas do Olho/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Fosfo-Específicos/imunologia , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Indução Embrionária , Olho/embriologia , Olho/enzimologia , Olho/metabolismo , Proteínas do Olho/química , Proteínas do Olho/genética , Regulação da Expressão Gênica , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Conformação Proteica , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Especificidade por Substrato , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The existence of vast regulatory networks mediated by microRNAs (miRNAs) suggests broad potential for miRNA dysfunction to contribute to disease. However, relatively few miRNA-target interactions are likely to make detectable contributions to phenotype, and effective strategies to identify these few interactions are currently wanting. We hypothesized that signaling cascades represent critical points of susceptibility to miRNA dysfunction, and we developed a strategy to test this theory by using quantitative cell-based screens. Here we report a screen for miRNAs that affect the Wingless (Wg) pathway, a conserved pathway that regulates growth and tissue specification. This process identified ectopic miR-315 as a potent and specific activator of Wg signaling, an activity that we corroborated in transgenic animals. This miR-315 activity was mediated by direct inhibition of Axin and Notum, which encode essential, negatively acting components of the Wg pathway. Genetic interaction tests substantiated both of these genes as key functional targets of miR-315. The ability of ectopic miR-315 to activate Wg signaling was not a trivial consequence of predicted miRNA-target relationships because other miRNAs with conserved sites in the Axin 3' UTR neither activated Wg outputs nor inhibited an Axin sensor. In summary, activity-based screening can selectively identify miRNAs whose deregulation can lead to interpretable phenotypic consequences.
Assuntos
Proteínas de Drosophila/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA , Drosophila , Proteína Wnt1RESUMO
KRAS, an oncogene mutated in nearly one third of human cancers, remains a pharmacologic challenge for direct inhibition except for recent advances in selective inhibitors targeting the G12C variant. Here, we report that selective inhibition of the protein tyrosine phosphatase, SHP2, can impair the proliferation of KRAS-mutant cancer cells in vitro and in vivo using cell line xenografts and primary human tumors. In vitro, sensitivity of KRAS-mutant cells toward the allosteric SHP2 inhibitor, SHP099, is not apparent when cells are grown on plastic in 2D monolayer, but is revealed when cells are grown as 3D multicellular spheroids. This antitumor activity is also observed in vivo in mouse models. Interrogation of the MAPK pathway in SHP099-treated KRAS-mutant cancer models demonstrated similar modulation of p-ERK and DUSP6 transcripts in 2D, 3D, and in vivo, suggesting a MAPK pathway-dependent mechanism and possible non-MAPK pathway-dependent mechanisms in tumor cells or tumor microenvironment for the in vivo efficacy. For the KRASG12C MIAPaCa-2 model, we demonstrate that the efficacy is cancer cell intrinsic as there is minimal antiangiogenic activity by SHP099, and the effects of SHP099 is recapitulated by genetic depletion of SHP2 in cancer cells. Furthermore, we demonstrate that SHP099 efficacy in KRAS-mutant models can be recapitulated with RTK inhibitors, suggesting RTK activity is responsible for the SHP2 activation. Taken together, these data reveal that many KRAS-mutant cancers depend on upstream signaling from RTK and SHP2, and provide a new therapeutic framework for treating KRAS-mutant cancers with SHP2 inhibitors.
Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Taquicininas/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias/patologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Post-translational modifications such as phosphorylation provide versatile context-specific strategies for modulating transcription factor activity. In the prevailing view of this process, modifying enzymes indirectly influence gene expression by shuttling to and from the nucleus where they alter the activity of their target transcription factors. However, a new paradigm has recently been suggested from studies of Eyes absent (EYA), a member of a conserved network of transcriptional regulators implicated in the development of numerous tissues and organs including the eye, ear, muscle and kidney. These findings indicate that EYA operates both as a transcriptional coactivator and as the prototype of a novel class of protein tyrosine phosphatases. The regulatory potential of such a bifunctional transcription factor is enormous and suggests a new layer of dynamic regulation in which transcription factors themselves might provide intrinsic enzymatic activities to fine-tune nuclear output.
Assuntos
Proteínas de Drosophila/fisiologia , Proteínas do Olho/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Fatores de Transcrição/fisiologia , Animais , Drosophila melanogaster , Regulação da Expressão Gênica/fisiologiaRESUMO
Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin. In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Esferoides Celulares/efeitos dos fármacos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Fibronectinas/metabolismo , HumanosRESUMO
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
The retinal determination (RD) gene network encodes a group of transcription factors and cofactors necessary for eye development. Transcriptional and posttranslational regulation of RD family members is achieved through interactions within the network and with extracellular signaling pathways, including epidermal growth factor receptor/RAS/mitogen-activated protein kinase (MAPK), transforming growth factor beta/DPP, Wingless, Hedgehog, and Notch. Here we present the results of structure-function analyses that reveal novel aspects of Eyes absent (EYA) function and regulation. We find that the conserved C-terminal EYA domain negatively regulates EYA transactivation potential, and that GROUCHO-SINE OCULIS (SO) interactions provide another mechanism for negative regulation of EYA-SO target genes. We have mapped the transactivation potential of EYA to an internal proline-, serine-, and threonine-rich region that includes the EYA domain 2 (ED2) and two MAPK phosphorylation consensus sites and demonstrate that activation of the RAS/MAPK pathway potentiates transcriptional output of EYA and the EYA-SO complex in certain contexts. Drosophila S2 cell two-hybrid assays were used to describe a novel homotypic interaction that is mediated by EYA's N terminus. Our data suggest that EYA requires homo- and heterotypic interactions and RAS/MAPK signaling responsiveness to ensure context-appropriate RD gene network activity.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Retina/fisiologia , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Estrutura Terciária de Proteína , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Eyes absent (EYA) proteins are defined by a conserved C-terminal EYA domain (ED) that both contributes to its function as a transcriptional coactivator by mediating protein-protein interactions and possesses intrinsic protein tyrosine phosphatase activity. Mutations in human EYA1 result in an autosomal dominant disorder called branchio-oto-renal (BOR) syndrome as well as congenital cataracts and ocular defects (OD). Both BOR- and OD-associated missense mutations alter residues in the conserved ED as do three missense mutations identified from Drosophila eya alleles. To investigate the molecular mechanisms whereby these mutations disrupt EYA function, we tested their activity in a series of assays that measured in vivo function, phosphatase activity, transcriptional capability, and protein-protein interactions. We find that the OD-associated mutations retain significant in vivo activity whereas those derived from BOR patients show a striking decrease or loss of in vivo functionality. Protein-protein interactions, either with its partner transcription factor Sine oculis or with EYA itself, were not significantly compromised. Finally, the results of the biochemical assays suggest that both loss of protein tyrosine phosphatase activity and reduced transcriptional capability contribute to the impaired EYA function associated with BOR/OD syndrome, thus shedding new light into the molecular mechanisms underlying this disease.
Assuntos
Síndrome Brânquio-Otorrenal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatases/genética , Transcrição Gênica , Alelos , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Drosophila/fisiologia , Proteínas do Olho/fisiologia , Genes Dominantes , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Cinética , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Proteínas Nucleares/fisiologia , Fenótipo , Células Fotorreceptoras de Invertebrados , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/fisiologia , Homologia de Sequência de Aminoácidos , Ativação Transcricional , TransgenesRESUMO
Glioblastoma (GBM) harbors subpopulations of therapy-resistant tumor-initiating cells (TICs) that are self-renewing and multipotent. To understand the regulation of the TIC state, we performed an image-based screen for genes regulating GBM TIC maintenance and identified ZFHX4, a 397 kDa transcription factor. ZFHX4 is required to maintain TIC-associated and normal human neural precursor cell phenotypes in vitro, suggesting that ZFHX4 regulates differentiation, and its suppression increases glioma-free survival in intracranial xenografts. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex. ZFHX4 and CHD4 bind to overlapping sets of genomic loci and control similar gene expression programs. Using expression data derived from GBM patients, we found that ZFHX4 significantly affects CHD4-mediated gene expression perturbations, which defines ZFHX4 as a master regulator of CHD4. These observations define ZFHX4 as a regulatory factor that links the chromatin-remodeling NuRD complex and the GBM TIC state.