RESUMEN
Genomic instability is a hallmark of cancer, and has a central role in the initiation and development of breast cancer1,2. The success of poly-ADP ribose polymerase inhibitors in the treatment of breast cancers that are deficient in homologous recombination exemplifies the utility of synthetically lethal genetic interactions in the treatment of breast cancers that are driven by genomic instability3. Given that defects in homologous recombination are present in only a subset of breast cancers, there is a need to identify additional driver mechanisms for genomic instability and targeted strategies to exploit these defects in the treatment of cancer. Here we show that centrosome depletion induces synthetic lethality in cancer cells that contain the 17q23 amplicon, a recurrent copy number aberration that defines about 9% of all primary breast cancer tumours and is associated with high levels of genomic instability4-6. Specifically, inhibition of polo-like kinase 4 (PLK4) using small molecules leads to centrosome depletion, which triggers mitotic catastrophe in cells that exhibit amplicon-directed overexpression of TRIM37. To explain this effect, we identify TRIM37 as a negative regulator of centrosomal pericentriolar material. In 17q23-amplified cells that lack centrosomes, increased levels of TRIM37 block the formation of foci that comprise pericentriolar material-these foci are structures with a microtubule-nucleating capacity that are required for successful cell division in the absence of centrosomes. Finally, we find that the overexpression of TRIM37 causes genomic instability by delaying centrosome maturation and separation at mitotic entry, and thereby increases the frequency of mitotic errors. Collectively, these findings highlight TRIM37-dependent genomic instability as a putative driver event in 17q23-amplified breast cancer and provide a rationale for the use of centrosome-targeting therapeutic agents in treating these cancers.
Asunto(s)
Neoplasias de la Mama/genética , Centrosoma/metabolismo , Centrosoma/patología , Cromosomas Humanos Par 17/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Centrosoma/efectos de los fármacos , Femenino , Fase G2 , Inestabilidad Genómica , Humanos , Mitosis/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Medios de Cultivo/química , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Western Blotting , Ciclo Celular/fisiología , Línea Celular , Técnicas de Cocultivo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Electrónica de Rastreo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Técnicas de Placa-Clamp , Receptores de GABA-A/metabolismoRESUMEN
Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABAA receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia.
Asunto(s)
Calcio/metabolismo , Diferenciación Celular/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Receptores de GABA-A/metabolismo , Animales , Humanos , Neurogénesis/fisiologíaRESUMEN
The metabolic properties of cancer cells have been widely accepted as a hallmark of cancer for a number of years and have shown to be of critical importance in tumour development. It is generally accepted that tumour cells exhibit a more glycolytic phenotype than normal cells. In this study, we investigate the bioenergetic phenotype of two widely used cancer cell lines, RD and U87MG, by monitoring intracellular oxygen concentrations using phosphorescent Pt-porphyrin based intracellular probes. Our study demonstrates that cancer cell lines do not always exhibit an exclusively glycolytic phenotype. RD demonstrates a reliance on oxidative phosphorylation whilst U87MG display a more glycolytic phenotype. Using the intracellular oxygen sensing probe we generate an immediate readout of intracellular oxygen levels, with the glycolytic lines reflecting the oxygen concentration of the environment, and cells with an oxidative phenotype having significantly lower levels of intracellular oxygen. Inhibition of oxygen consumption in lines with high oxygen consumption increases intracellular oxygen levels towards environmental levels. We conclude that the use of intracellular oxygen probes provides a quantitative assessment of intracellular oxygen levels, allowing the manipulation of cellular bioenergetics to be studied in real time.
Asunto(s)
Oxígeno/análisis , Línea Celular Tumoral , Metabolismo Energético , Glucólisis , Humanos , Consumo de OxígenoRESUMEN
Inappropriate osteoclast activity instigates pathological bone loss in rheumatoid arthritis. We have investigated how osteoclasts generate sufficient ATP for the energy-intensive process of bone resorption in the hypoxic microenvironment associated with this rheumatic condition. We show that in human osteoclasts differentiated from CD14(+) monocytes, hypoxia (24 h, 2% O2 ): (a) increases ATP production and mitochondrial electron transport chain activity (Alamar blue, O2 consumption); (b) increases glycolytic flux (glucose consumption, lactate production); and (c) increases glutamine consumption. We demonstrate that glucose, rather than glutamine, is necessary for the hypoxic increase in ATP production and also for cell survival in hypoxia. Using siRNA targeting specific isoforms of the hypoxia-inducible transcription factor HIF (HIF-1α, HIF-2α), we show that employment of selected components of the HIF-1α-mediated metabolic switch to anaerobic respiration enables osteoclasts to rapidly increase ATP production in hypoxia, while at the same time compromising long-term survival. We propose this atypical HIF-driven metabolic pathway to be an adaptive mechanism to permit rapid bone resorption in the short term while ensuring curtailment of the process in the absence of re-oxygenation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Resorción Ósea/metabolismo , Metabolismo Energético , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mitocondrias/metabolismo , Osteoclastos/metabolismo , Adaptación Fisiológica , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Resorción Ósea/genética , Resorción Ósea/patología , Resorción Ósea/fisiopatología , Hipoxia de la Célula , Supervivencia Celular , Células Cultivadas , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Glucosa/metabolismo , Glutamina/metabolismo , Glucólisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ácido Láctico/metabolismo , Osteoclastos/patología , Consumo de Oxígeno , Complejo Piruvato Deshidrogenasa/metabolismo , Interferencia de ARN , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
Adenoviruses (Ads) have demonstrated significant success as replication-deficient (RD) viral vectored vaccines, as well as broad potential across gene therapy and cancer therapy. Ad vectors transduce human cells via direct interactions between the viral fiber knob and cell surface receptors, with secondary cellular integrin interactions. Ad receptor usage is diverse across the extensive phylogeny. Commonly studied human Ad serotype 5 (Ad5), and chimpanzee Ad-derived vector "ChAdOx1" in licensed ChAdOx1 nCoV-19 vaccine, both form primary interactions with the coxsackie and adenovirus receptor (CAR), which is expressed on human epithelial cells and erythrocytes. CAR usage is suboptimal for targeted gene delivery to cells with low/negative CAR expression, including human dendritic cells (DCs) and vascular smooth muscle cells (VSMCs). We evaluated the performance of an RD Ad5 vector pseudotyped with the fiber knob of human Ad serotype 49, termed Ad5/49K vector. Ad5/49K demonstrated superior transduction of murine and human DCs over Ad5, which translated into significantly increased T cell immunogenicity when evaluated in a mouse cancer vaccine model using 5T4 tumor-associated antigen. Additionally, Ad5/49K exhibited enhanced transduction of primary human VSMCs. These data highlight the potential of Ad5/49K vector for both vascular gene therapy applications and as a potent vaccine vector.
RESUMEN
Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors.
Asunto(s)
Cromatina/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteína que Contiene Valosina/metabolismo , Línea Celular Tumoral , Disulfiram/análogos & derivados , Disulfiram/farmacología , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Sumoilación , Factores de Transcripción/metabolismo , UbiquitinaciónRESUMEN
We previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvß6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvß6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvß6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvß6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvß6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvß6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvß6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes.
Asunto(s)
Adenoviridae/genética , Antígenos de Neoplasias/genética , Terapia Genética , Vectores Genéticos/genética , Integrinas/genética , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Administración Intravenosa , Animales , Línea Celular Tumoral , Supervivencia Celular , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Humanos , Ratones , Neoplasias/etiología , Viroterapia Oncolítica/métodos , Transducción Genética , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Reparación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Mutaciones Letales Sintéticas/efectos de los fármacos , Regulación Alostérica , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleasas/antagonistas & inhibidores , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Recombinación Homóloga/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Organoides/efectos de los fármacos , Neoplasias Ováricas/genética , Ratas , Mutaciones Letales Sintéticas/genética , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , ADN Polimerasa thetaRESUMEN
Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Organoides/efectos de los fármacos , Tanquirasas/antagonistas & inhibidores , Adulto , Animales , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/patología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Organoides/patologíaRESUMEN
Purpose: Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy.Experimental Design: We evaluated FRα expression in breast cancers by genomic (n = 3,414) and IHC (n = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the antitumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro, and in human TNBC xenograft models.Results: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in postneoadjuvant chemotherapy-residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1, and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth.Conclusions: FRα is overexpressed in high-grade TNBC and postchemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as ADCs, or passive immunotherapy priming Fc-mediated antitumor immune cell responses. Clin Cancer Res; 24(20); 5098-111. ©2018 AACR.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Receptor 1 de Folato/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/efectos adversos , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Femenino , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Modelos Biológicos , Terapia Molecular Dirigida , Neoplasias Basocelulares , Interferencia de ARN , Transducción de Señal , Neoplasias de la Mama Triple Negativas/patología , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Background: Mitochondrial diabetes is primarily caused by ß-cell failure, a cell type whose unique properties are important in pathogenesis. Methods: By reducing glucose, we induced energetic stress in two rodent ß-cell models to assess effects on cellular function. Results: Culturing rat insulin-secreting INS-1 cells in low glucose conditions caused a rapid reduction in whole cell respiration, associated with elevated mitochondrial reactive oxygen species production, and an altered glucose-stimulated insulin secretion profile. Prolonged exposure to reduced glucose directly impaired mitochondrial function and reduced autophagy. Conclusions: Insulinoma cell lines have a very different bioenergetic profile to many other cell lines and provide a useful model of mechanisms affecting ß-cell mitochondrial function.