RESUMEN
MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proliferación Celular/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , Proteínas Represoras/metabolismo , Aptámeros de Nucleótidos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Proteína 9 Asociada a CRISPR/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Regiones Promotoras Genéticas , Dominios Proteicos , ARN Guía de Kinetoplastida , ARN Largo no Codificante/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Luciferasas de Renilla , Ratones , Fosforilación , Pez CebraRESUMEN
The MYC protein is a transcription factor with oncogenic potential controlling fundamental cellular processes such as cell proliferation, metabolism, differentiation, and apoptosis. The MYC gene is a major cancer driver, and elevated MYC protein levels are a hallmark of most human cancers. We have previously shown that the brain acid-soluble protein 1 gene (BASP1) is specifically downregulated by the v-myc oncogene and that ectopic BASP1 expression inhibits v-myc-induced cell transformation. The 11-amino acid effector domain of the BASP1 protein interacts with the calcium sensor calmodulin (CaM) and is mainly responsible for this inhibitory function. We also reported recently that CaM interacts with all MYC variant proteins and that ectopic CaM increases the transactivation and transformation potential of the v-Myc protein. Here, we show that the presence of excess BASP1 or of a synthetic BASP1 effector domain peptide leads to displacement of v-Myc from CaM. The protein stability of v-Myc is decreased in cells co-expressing v-Myc and BASP1, which may account for the inhibition of v-Myc. Furthermore, suppression of v-Myc-triggered transcriptional activation and cell transformation is compensated by ectopic CaM, suggesting that BASP1-mediated withdrawal of CaM from v-Myc is a crucial event in the inhibition. In view of the tumor-suppressive role of BASP1 which was recently also reported for human cancer, small compounds or peptides based on the BASP1 effector domain could be used in drug development strategies aimed at tumors with high MYC expression.
Asunto(s)
Calmodulina/metabolismo , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Calmodulina/antagonistas & inhibidores , Calmodulina/genética , Línea Celular Tumoral , Proliferación Celular/genética , Células Cultivadas , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Codorniz , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tumor formation is generally linked to the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed or contain a single or double knock-in (DKI) of PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC target gene signature. This gene signature was mediated in part by the bromodomain-containing protein 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced MYC transcript levels. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR-Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and BRD9 overexpression induced the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncoprotein cooperation and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS-driven oncogenic transformation.
RESUMEN
Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation-containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.
Asunto(s)
Melanoma/tratamiento farmacológico , Melanoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Antineoplásicos/farmacología , Técnicas Biosensibles , Dominio Catalítico/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/genética , Conformación Molecular , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de SeñalRESUMEN
Recently, the palbociclib/letrozole combination therapy was granted accelerated US FDA approval for the treatment of estrogen receptor (ER)-positive breast cancer. Since the underlying metabolic effects of these drugs are yet unknown, we investigated their synergism at the metabolome level in MCF-7 cells. As xenoestrogens interact with the ER, we additionally aimed at deciphering the impact of the phytoestrogen genistein and the estrogenic mycotoxin zearalenone. A global metabolomics approach was applied to unravel metabolite and pathway modifications. The results clearly showed that the combined effects of palbociclib and letrozole on cellular metabolism were far more pronounced than that of each agent alone and potently influenced by xenoestrogens. This behavior was confirmed in proliferation experiments and functional assays. Specifically, amino acids and central carbon metabolites were attenuated, while higher abundances were observed for fatty acids and most nucleic acid-related metabolites. Interestingly, exposure to model xenoestrogens appeared to counteract these effects.
Asunto(s)
Letrozol/farmacología , Metaboloma/efectos de los fármacos , Fitoestrógenos/farmacología , Piperazinas/farmacología , Piridinas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Carbono/metabolismo , Dieta , Femenino , Genisteína/química , Genisteína/farmacología , Humanos , Letrozol/química , Letrozol/uso terapéutico , Células MCF-7 , Metabolómica , Fitoestrógenos/química , Piperazinas/química , Piperazinas/uso terapéutico , Análisis de Componente Principal , Piridinas/química , Piridinas/uso terapéutico , Receptores de Estrógenos/metabolismo , Zearalenona/química , Zearalenona/farmacologíaRESUMEN
The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencia de Aminoácidos , Animales , Calmodulina/química , Línea Celular , Humanos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Recombinantes de Fusión/metabolismo , Activación TranscripcionalRESUMEN
Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Mapeo de Interacción de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Animales , Técnicas Biosensibles , Línea Celular Tumoral , Embrión no Mamífero/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Ratones , Osteosarcoma/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
The oncogenic bHLH-LZ transcription factor Myc forms binary complexes with its binding partner Max. These and other bHLH-LZ-based protein-protein interactions (PPI) in the Myc-Max network are essential for the physiological and oncogenic activities of Myc. We have generated a genetically determined and highly specific protein-fragment complementation assay based on Renilla luciferase to analyze the dynamic interplay of bHLH-LZ transcription factors Myc, Max, and Mxd1 in vivo. We also applied this PPI reporter to quantify alterations of nuclear Myc-Max complexes in response to mutational events, competitive binding by the transcriptional repressor Mxd1, or perturbations by small-molecule Myc inhibitors, including recently identified potent PPI inhibitors from a Kröhnke pyridine library. We show that the specificity of Myc-Max PPI reduction by the pyridine inhibitors directly correlates with their efficient and highly specific potential to interfere with the proliferation of human and avian tumor cells displaying deregulated Myc expression. In a direct comparison with known Myc inhibitors using human and avian cell systems, the pyridine compounds reveal a unique inhibitory potential even at sub-micromolar concentrations combined with remarkable specificity for the inhibition of Myc-driven tumor cell proliferation. Furthermore, we show in direct comparisons using defined avian cell systems that different Max PPI profiles for the variant members of the Myc protein family (c-Myc, v-Myc, N-Myc, L-Myc) correlate with their diverse oncogenic potential and their variable sensitivity to the novel pyridine inhibitors.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HEK293 , Humanos , Luciferasas de Renilla , Neoplasias/metabolismo , Oxadiazoles/farmacología , Unión Proteica , Multimerización de Proteína , Piridinas/farmacología , Tiazoles/farmacologíaRESUMEN
The c-myc protooncogene encodes the Myc transcription factor, a global regulator of fundamental cellular processes. Deregulation of c-myc leads to tumorigenesis, and c-myc is an important driver in human cancer. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins involved in transcriptional regulation of target genes. Non-transcriptional functions have also been attributed to the Myc protein, notably direct interaction with the pre-replicative complex (pre-RC) controlling the initiation of DNA replication. A key component of the pre-RC is the Cdt1 protein, an essential factor in origin licensing. Here we present data suggesting that the CDT1 gene is a transcriptional target of the Myc-Max complex. Expression of the CDT1 gene in v-myc-transformed cells directly correlates with myc expression. Also, human tumor cells with elevated c-myc expression display increased CDT1 expression. Occupation of the CDT1 promoter by Myc-Max is demonstrated by chromatin immunoprecipitation, and transactivation by Myc-Max is shown in reporter assays. Ectopic expression of CDT1 leads to cell transformation. Our results provide a possible direct mechanistic link of Myc's canonical function as a transcription factor to DNA replication. Furthermore, we suggest that aberrant transcriptional activation of CDT1 by deregulated myc alleles contributes to the genomic instabilities observed in tumor cells.