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1.
Science ; 383(6690): 1441-1448, 2024 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-38547292

RESUMEN

Mitotic duration is tightly constrained, and extended mitosis is characteristic of problematic cells prone to chromosome missegregation and genomic instability. We show here that mitotic extension leads to the formation of p53-binding protein 1 (53BP1)-ubiquitin-specific protease 28 (USP28)-p53 protein complexes that are transmitted to, and stably retained by, daughter cells. Complexes assembled through a Polo-like kinase 1-dependent mechanism during extended mitosis and elicited a p53 response in G1 that prevented the proliferation of the progeny of cells that experienced an approximately threefold extended mitosis or successive less extended mitoses. The ability to monitor mitotic extension was lost in p53-mutant cancers and some p53-wild-type (p53-WT) cancers, consistent with classification of TP53BP1 and USP28 as tumor suppressors. Cancers retaining the ability to monitor mitotic extension exhibited sensitivity to antimitotic agents.


Asunto(s)
Proliferación Celular , Mitosis , Neoplasias , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina Tiolesterasa , Humanos , Proliferación Celular/genética , Inestabilidad Genómica , Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias/genética , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Línea Celular Tumoral , Quinasa Tipo Polo 1/metabolismo , Antimitóticos/farmacología , Resistencia a Antineoplásicos
2.
Cancers (Basel) ; 14(23)2022 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-36497283

RESUMEN

Breast cancer is a common cancer in women. Breast cancer cells synthesize large amounts of hyaluronan to assist their proliferation, survival, migration and invasion. Accumulation of hyaluronan and overexpression of its receptor CD44 and hyaluronidase TMEM2 in breast tumors correlate with tumor progression and reduced overall survival of patients. Currently, the only known small molecule inhibitor of hyaluronan synthesis is 4-methyl-umbelliferone (4-MU). Due to the importance of hyaluronan for breast cancer progression, our aim was to identify new, potent and chemically distinct inhibitors of its synthesis. Here, we report a new small molecule inhibitor of hyaluronan synthesis, the thymidine analog 5'-Deoxy-5'-(1,3-Diphenyl-2-Imidazolidinyl)-Thymidine (DDIT). This compound is more potent than 4-MU and displays significant anti-tumorigenic properties. Specifically, DDIT inhibits breast cancer cell proliferation, migration, invasion and cancer stem cell self-renewal by suppressing HAS-synthesized hyaluronan. DDIT appears as a promising lead compound for the development of inhibitors of hyaluronan synthesis with potential usefulness in breast cancer treatment.

3.
Mol Biol Cell ; 32(12): 1193-1201, 2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-33852350

RESUMEN

Centromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of Caenorhabditis elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-tail containing the predicted structured region binds to KNL-2, a conserved SANTA domain and Myb domain-containing protein (referred to as M18BP1 in vertebrates) specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode Caenorhabditis briggsae, despite divergence of the N-tail and KNL-2 primary sequences. Thus, the extended N-tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates a direct interaction between CENP-A and KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Caenorhabditis elegans/genética , Proteína A Centromérica/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos
4.
Nat Commun ; 12(1): 1337, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33637727

RESUMEN

Identification of the cis-regulatory elements controlling cell-type specific gene expression patterns is essential for understanding the origin of cellular diversity. Conventional assays to map regulatory elements via open chromatin analysis of primary tissues is hindered by sample heterogeneity. Single cell analysis of accessible chromatin (scATAC-seq) can overcome this limitation. However, the high-level noise of each single cell profile and the large volume of data pose unique computational challenges. Here, we introduce SnapATAC, a software package for analyzing scATAC-seq datasets. SnapATAC dissects cellular heterogeneity in an unbiased manner and map the trajectories of cellular states. Using the Nyström method, SnapATAC can process data from up to a million cells. Furthermore, SnapATAC incorporates existing tools into a comprehensive package for analyzing single cell ATAC-seq dataset. As demonstration of its utility, SnapATAC is applied to 55,592 single-nucleus ATAC-seq profiles from the mouse secondary motor cortex. The analysis reveals ~370,000 candidate regulatory elements in 31 distinct cell populations in this brain region and inferred candidate cell-type specific transcriptional regulators.


Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina/métodos , Análisis de la Célula Individual/métodos , Animales , Cromatina , Biología Computacional , Epigenómica , Masculino , Ratones , Ratones Endogámicos C57BL , Corteza Motora , Análisis de Secuencia de ADN/métodos
5.
J Cell Biol ; 219(12)2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-33170211

RESUMEN

Centrosomes, composed of centrioles that recruit a pericentriolar material (PCM) matrix assembled from PCNT and CDK5RAP2, catalyze mitotic spindle assembly. Here, we inhibit centriole formation and/or remove PCNT-CDK5RAP2 in RPE1 cells to address their relative contributions to spindle formation. While CDK5RAP2 and PCNT are normally dispensable for spindle formation, they become essential when centrioles are absent. Acentriolar spindle assembly is accompanied by the formation of foci containing PCNT and CDK5RAP2 via a microtubule and Polo-like kinase 1-dependent process. Foci formation and spindle assembly require PCNT-CDK5RAP2-dependent matrix assembly and the ability of CDK5RAP2 to recruit γ-tubulin complexes. Thus, the PCM matrix can self-organize independently of centrioles to generate microtubules for spindle assembly; conversely, an alternative centriole-anchored mechanism supports spindle assembly when the PCM matrix is absent. Extension to three cancer cell lines revealed similar results in HeLa cells, whereas DLD1 and U2OS cells could assemble spindles in the absence of centrioles and PCNT-CDK5RAP2, suggesting cell type variation in spindle assembly mechanisms.


Asunto(s)
Antígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Antígenos/genética , Proteínas de Ciclo Celular/genética , Centriolos/genética , Células HeLa , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Quinasa Tipo Polo 1
6.
Nature ; 585(7825): 440-446, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32908304

RESUMEN

Centrosomes catalyse the formation of microtubules needed to assemble the mitotic spindle apparatus1. Centrosomes themselves duplicate once per cell cycle, in a process that is controlled by the serine/threonine protein kinase PLK4 (refs. 2,3). When PLK4 is chemically inhibited, cell division proceeds without centrosome duplication, generating centrosome-less cells that exhibit delayed, acentrosomal spindle assembly4. Whether PLK4 inhibitors can be leveraged as a treatment for cancer is not yet clear. Here we show that acentrosomal spindle assembly following PLK4 inhibition depends on levels of the centrosomal ubiquitin ligase TRIM37. Low TRIM37 levels accelerate acentrosomal spindle assembly and improve proliferation following PLK4 inhibition, whereas high TRIM37 levels inhibit acentrosomal spindle assembly, leading to mitotic failure and cessation of proliferation. The Chr17q region containing the TRIM37 gene is frequently amplified in neuroblastoma and in breast cancer5-8, rendering these cancer types highly sensitive to PLK4 inhibition. We find that inactivating TRIM37 improves acentrosomal mitosis because TRIM37 prevents PLK4 from self-assembling into centrosome-independent condensates that serve as ectopic microtubule-organizing centres. By contrast, elevated TRIM37 expression inhibits acentrosomal spindle assembly through a distinct mechanism that involves degradation of the centrosomal component CEP192. Thus, TRIM37 is an essential determinant of mitotic vulnerability to PLK4 inhibition. Linkage of TRIM37 to prevalent cancer-associated genomic changes-including 17q gain in neuroblastoma and 17q23 amplification in breast cancer-may offer an opportunity to use PLK4 inhibition to trigger selective mitotic failure and provide new avenues to treatments for these cancers.


Asunto(s)
Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos Par 17/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Centro Organizador de los Microtúbulos/efectos de los fármacos , Centro Organizador de los Microtúbulos/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Sulfonas/farmacología , Sulfonas/uso terapéutico , Ubiquitina/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Curr Biol ; 30(16): 3101-3115.e11, 2020 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-32619481

RESUMEN

Cytokinesis partitions the cell contents to complete mitosis. During cytokinesis, polo-like kinase 1 (PLK1) activates the small GTPase RhoA to assemble a contractile actomyosin ring. PLK1 is proposed to pattern RhoA activation by creating a docking site on the central spindle that concentrates the RhoA guanine nucleotide exchange factor ECT2. However, ECT2 targeting to the central spindle is dispensable for cytokinesis, indicating that how PLK1 controls RhoA activation remains unresolved. To address this question, we employed an unbiased approach targeting ∼100 predicted PLK1 sites in two RhoA regulators: ECT2 and the centralspindlin complex, composed of CYK4 and kinesin-6. This comprehensive approach suggested that the only functionally critical PLK1 target sites are in a single cluster in the CYK4 N terminus. Phosphorylation of this cluster promoted direct interaction of CYK4 with the BRCT repeat module of ECT2. However, mutational analysis in vitro and in vivo led to the surprising finding that the interaction was independent of the conserved "canonical" residues in ECT2's BRCT repeat module that, based on structurally characterized BRCT-phosphopeptide interactions, were presumed critical for binding. Instead, we show that the ECT2 BRCT module binds phosphorylated CYK4 via a distinct conserved basic surface. Basic surface mutations mimic the effects on cytokinesis of loss of CYK4 cluster phosphorylation or inhibition of PLK1 activity. Together with evidence for ECT2 autoinhibition limiting interaction with CYK4 in the cytoplasm, these results suggest that a spatial gradient of phosphorylated CYK4 around the central spindle patterns RhoA activation by interacting with ECT2 on the adjacent plasma membrane.


Asunto(s)
Proteína BRCA1/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Citocinesis , Fosfopéptidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Proteína BRCA1/genética , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Fosfopéptidos/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Huso Acromático , Proteína de Unión al GTP rhoA/genética , Quinasa Tipo Polo 1
10.
Cancer Cell ; 35(3): 504-518.e7, 2019 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-30827889

RESUMEN

Ionizing radiation (IR) and chemotherapy are standard-of-care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knockin mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.


Asunto(s)
Neoplasias Encefálicas/terapia , Núcleo Celular/metabolismo , Glioma/terapia , Fosfohidrolasa PTEN/metabolismo , Pirimidinas/administración & dosificación , Tolerancia a Radiación/efectos de los fármacos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Reparación del ADN/efectos de los fármacos , Femenino , Glioma/metabolismo , Humanos , Masculino , Ratones , Fosforilación/efectos de los fármacos , Pirimidinas/farmacología , Recombinasa Rad51/metabolismo , Tirosina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
11.
J Allergy Clin Immunol ; 144(1): 224-235, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30707971

RESUMEN

BACKGROUND: Janus kinase (JAK) 2 plays pivotal roles in signaling by several cytokine receptors. The mutant JAK2 V617F is the most common molecular event associated with myeloproliferative neoplasms. Selective targeting of the mutant would be ideal for treating these pathologies by sparing essential JAK2 functions. OBJECTIVE: We characterize inhibitory strategies for JAK2 V617F and assess their effect on physiologic signaling by distinct cytokine receptors. METHODS: Through structure-guided mutagenesis, we assessed the role of key residues around F617 and used a combination of cellular and biochemical assays to measure the activity of JAKs in reconstituted cells. We also assessed the effect of several specific JAK2 V617F inhibitory mutations on receptor dimerization using the NanoBiT protein complementation approach. RESULTS: We identified a novel Janus kinase homology 2 (JH2) αC mutation, A598F, which is suggested to inhibit the aromatic stacking between F617 with F594 and F595. Like other JAK2 V617F inhibitory mutations, A598F decreased oncogenic activation and spared cytokine activation while preventing JAK2 V617F-promoted erythropoietin receptor dimerization. Surprisingly, A598F and other V617F-inhibiting mutations (F595A, E596R, and F537A) significantly impaired IFN-γ signaling. This was specific for IFN-γ because the inhibitory mutations preserved responses to ligands of a series of receptor complexes. Similarly, homologous mutations in JAK1 prevented signaling by IFN-γ. CONCLUSIONS: The JH2 αC region, which is required for JAK2 V617F hyperactivation, is crucial for relaying cytokine-induced signaling of the IFN-γ receptor. We discuss how strategies aiming to inhibit JAK2 V617F could be used for identifying inhibitors of IFN-γ signaling.


Asunto(s)
Janus Quinasa 2/genética , Receptores de Citocinas/metabolismo , Animales , Línea Celular , Humanos , Janus Quinasa 2/metabolismo , Ratones , Mutación , Transducción de Señal
13.
Methods Mol Biol ; 1787: 53-66, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29736709

RESUMEN

An inhibitor for the thrombopoietin receptor (TpoR) would be more specific for the treatment of myeloproliferative neoplasms (MPNs) due to constitutively active mutant TpoR compared to the current treatment approach of inhibiting Janus kinase 2 (JAK2). We describe a cell-based high-throughput phenotypic screening approach to identify inhibitors for constitutively active mutant TpoR. A stepwise elimination process is used to differentiate generally cytotoxic compounds from compounds that specifically inhibit growth of cells expressing wild-type TpoR and/or mutant TpoR. We have systematically optimized the phenotypic screening assay and documented in this chapter critical parameters for a successful phenotypic screen, such as cell growth and seeding optimization, plate reproducibility and uniformity studies, and an assay robustness analysis with a pilot screen.


Asunto(s)
Descubrimiento de Drogas , Fenotipo , Receptores de Trombopoyetina/antagonistas & inhibidores , Receptores de Trombopoyetina/genética , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Ligandos , Mediciones Luminiscentes/métodos , Ratones , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas
14.
Nat Genet ; 50(1): 73-82, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29255264

RESUMEN

Enhancers act to regulate cell-type-specific gene expression by facilitating the transcription of target genes. In mammalian cells, active or primed enhancers are commonly marked by monomethylation of histone H3 at lysine 4 (H3K4me1) in a cell-type-specific manner. Whether and how this histone modification regulates enhancer-dependent transcription programs in mammals is unclear. In this study, we conducted SILAC mass spectrometry experiments with mononucleosomes and identified multiple H3K4me1-associated proteins, including many involved in chromatin remodeling. We demonstrate that H3K4me1 augments association of the chromatin-remodeling complex BAF to enhancers in vivo and that, in vitro, H3K4me1-marked nucleosomes are more efficiently remodeled by the BAF complex. Crystal structures of the BAF component BAF45C indicate that monomethylation, but not trimethylation, is accommodated by BAF45C's H3K4-binding site. Our results suggest that H3K4me1 has an active role at enhancers by facilitating binding of the BAF complex and possibly other chromatin regulators.


Asunto(s)
Elementos de Facilitación Genéticos , Código de Histonas , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Células HeLa , Histonas/química , Humanos , Lisina/metabolismo , Ratones , Proteínas Nucleares/química , Nucleosomas/metabolismo , Unión Proteica
15.
Genes Dev ; 31(12): 1212-1227, 2017 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-28724615

RESUMEN

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.


Asunto(s)
Resistencia a Antineoplásicos/genética , Glioblastoma/fisiopatología , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal/genética , Animales , Comunicación Celular , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Interleucina-6/metabolismo , Ratones , Ratones Desnudos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
16.
Mol Cell ; 67(1): 128-138.e7, 2017 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-28648777

RESUMEN

Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic inhibition of the mammalian target of rapamycin (mTOR) kinase, promotes glutamate secretion, cystine uptake, and incorporation into glutathione, linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.


Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Neoplasias Encefálicas/enzimología , Cisteína/metabolismo , Glioblastoma/enzimología , Glutamina/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Células A549 , Sistema de Transporte de Aminoácidos y+/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glioblastoma/genética , Glioblastoma/patología , Glutatión/biosíntesis , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , Mutación , Fosforilación , Unión Proteica , Proteómica/métodos , Interferencia de ARN , Serina , Serina-Treonina Quinasas TOR/genética , Espectrometría de Masas en Tándem , Factores de Tiempo , Transfección , Microambiente Tumoral
17.
Mol Biol Cell ; 27(24): 3828-3840, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27708138

RESUMEN

The mechanism of directional cell migration remains an important problem, with relevance to cancer invasion and metastasis. GOLPH3 is a common oncogenic driver of human cancers, and is the first oncogene that functions at the Golgi in trafficking to the plasma membrane. Overexpression of GOLPH3 is reported to drive enhanced cell migration. Here we show that the phosphatidylinositol-4-phosphate/GOLPH3/myosin 18A/F-actin pathway that is critical for Golgi-to-plasma membrane trafficking is necessary and limiting for directional cell migration. By linking the Golgi to the actin cytoskeleton, GOLPH3 promotes reorientation of the Golgi toward the leading edge. GOLPH3 also promotes reorientation of lysosomes (but not other organelles) toward the leading edge. However, lysosome function is dispensable for migration and the GOLPH3 dependence of lysosome movement is indirect, via GOLPH3's effect on the Golgi. By driving reorientation of the Golgi to the leading edge and driving forward trafficking, particularly to the leading edge, overexpression of GOLPH3 drives trafficking to the leading edge of the cell, which is functionally important for directional cell migration. Our identification of a novel pathway for Golgi reorientation controlled by GOLPH3 provides new insight into the mechanism of directional cell migration with important implications for understanding GOLPH3's role in cancer.


Asunto(s)
Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Técnicas de Cultivo de Célula , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Polaridad Celular , Citoesqueleto , Células HeLa , Humanos , Proteínas de la Membrana/fisiología , Miosinas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de Proteínas/fisiología , Transducción de Señal
18.
Cancer Cell ; 30(5): 683-693, 2016 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-27746144

RESUMEN

Small-molecule inhibitors targeting growth factor receptors have failed to show efficacy for brain cancers, potentially due to their inability to achieve sufficient drug levels in the CNS. Targeting non-oncogene tumor co-dependencies provides an alternative approach, particularly if drugs with high brain penetration can be identified. Here we demonstrate that the highly lethal brain cancer glioblastoma (GBM) is remarkably dependent on cholesterol for survival, rendering these tumors sensitive to Liver X receptor (LXR) agonist-dependent cell death. We show that LXR-623, a clinically viable, highly brain-penetrant LXRα-partial/LXRß-full agonist selectively kills GBM cells in an LXRß- and cholesterol-dependent fashion, causing tumor regression and prolonged survival in mouse models. Thus, a metabolic co-dependency provides a pharmacological means to kill growth factor-activated cancers in the CNS.


Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Colesterol/metabolismo , Glioblastoma/tratamiento farmacológico , Indazoles/administración & dosificación , Receptores X del Hígado/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Glioblastoma/metabolismo , Humanos , Indazoles/farmacología , Ratones , Resultado del Tratamiento
19.
Sci Rep ; 6: 29868, 2016 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-27430378

RESUMEN

Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor ß (TGF-ß)-induced epithelial-mesenchymal transition. In addition to TGF-ß receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-ß-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-ß-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer.


Asunto(s)
Diferenciación Celular/genética , Receptores X del Hígado/genética , Miofibroblastos/citología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Diferenciación Celular/efectos de los fármacos , Colágeno/metabolismo , Desarrollo Embrionario/genética , Células Epiteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos/efectos de los fármacos , Receptores X del Hígado/agonistas , Ratones , Receptores de Factores de Crecimiento Transformadores beta , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Crecimiento Transformador beta/genética
20.
J Cell Biol ; 214(2): 155-66, 2016 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-27432897

RESUMEN

In normal human cells, centrosome loss induced by centrinone-a specific centrosome duplication inhibitor-leads to irreversible, p53-dependent G1 arrest by an unknown mechanism. A genome-wide CRISPR/Cas9 screen for centrinone resistance identified genes encoding the p53-binding protein 53BP1, the deubiquitinase USP28, and the ubiquitin ligase TRIM37. Deletion of TP53BP1, USP28, or TRIM37 prevented p53 elevation in response to centrosome loss but did not affect cytokinesis failure-induced arrest or p53 elevation after doxorubicin-induced DNA damage. Deletion of TP53BP1 and USP28, but not TRIM37, prevented growth arrest in response to prolonged mitotic duration. TRIM37 knockout cells formed ectopic centrosomal-component foci that suppressed mitotic defects associated with centrosome loss. TP53BP1 and USP28 knockouts exhibited compromised proliferation after centrosome removal, suggesting that centrosome-independent proliferation is not conferred solely by the inability to sense centrosome loss. Thus, analysis of centrinone resistance identified a 53BP1-USP28 module as critical for communicating mitotic challenges to the p53 circuit and TRIM37 as an enforcer of the singularity of centrosome assembly.


Asunto(s)
Centrosoma/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Mitosis , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Biomarcadores/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Centrosoma/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Eliminación de Gen , Técnicas de Inactivación de Genes , Pruebas Genéticas , Humanos , Mitosis/efectos de los fármacos , Mutación/genética , Proteínas Nucleares/metabolismo , Pirimidinas/farmacología , Sulfonas/farmacología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas
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