RESUMO
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.
Assuntos
Neoplasias da Mama/genética , Centrossomo/metabolismo , Centrossomo/patologia , Cromossomos Humanos Par 17/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Centrossomo/efeitos dos fármacos , Feminino , Fase G2 , Instabilidade Genômica , Humanos , Mitose/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
OBJECTIVES: Synovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/ß-catenin signalling, given its emerging importance in arthritis. METHODS: We subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain of function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. RESULTS: We uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/ß-catenin signalling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages and chondrocytes. CONCLUSIONS: Synovial fibroblasts assume distinct functional identities during PTOA in mice, and Prg4hi lining fibroblasts secrete Rspo2 that may drive pathological joint crosstalk after injury.
Assuntos
Osteoartrite , Trombospondinas , Animais , Humanos , Camundongos , Condrócitos/metabolismo , Fibroblastos/metabolismo , Osteoartrite/patologia , Membrana Sinovial/metabolismo , Via de Sinalização Wnt , Trombospondinas/metabolismoRESUMO
Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co-culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue-like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two-dimensional (2D) substrates and review the popular approaches currently available that enable the development of three-dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold-free or scaffold-based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science.
Assuntos
Técnicas de Cultura de Células , Técnicas de Cocultura , Células-Tronco , Engenharia Tecidual/métodos , Alternativas aos Testes com Animais , Animais , Proliferação de Células , Humanos , Esferoides Celulares , Alicerces TeciduaisRESUMO
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.
Assuntos
Cromatina/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína com Valosina/metabolismo , Linhagem Celular Tumoral , Dissulfiram/análogos & derivados , Dissulfiram/farmacologia , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Neoplasias/tratamento farmacológico , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , UbiquitinaçãoRESUMO
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.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais/efeitos dos fármacos , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Recombinação Homóloga/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Organoides/efeitos dos fármacos , Neoplasias Ovarianas/genética , Ratos , Mutações Sintéticas Letais/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , DNA Polimerase tetaRESUMO
Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo-like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. SIGNIFICANCE: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.
Assuntos
Proteína de Ligação a CREB/fisiologia , Carcinogênese/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Animais , Proteína de Ligação a CREB/genética , Proliferação de Células/genética , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Genômica/métodos , Células HCT116 , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Invasividade Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIMS: The Scottish Transfusion and Laboratory Support in Trauma Group (TLSTG) previously reviewed all National Code Red activations between June 1st 2013 and October 31st 2015, generating a number of recommendations to be adopted to optimise the transfusion support given to patients following major trauma in Scotland. A repeat National survey was undertaken for all patients for whom Code Red was activated between 1st November 2015 and 31st December 2017. METHODS: A clinical and transfusion lead for each centre entered anonymised data onto a secure electronic database (REDCap). RESULTS: During the study period there were 66 activations (24 South-East of Scotland, 32 West, 10 East). Mean age was 45 years and 88% were male. Mean Injury Severity Score (ISS) was 28 with 75% blunt trauma. 93% (62/66) of Code Red patients received blood components with a 300% increase in pre-hospital transfusion (48 vs 16 patients; p<0.001). Median time from 999 call to Code Red activation reduced significantly to 37 min from 70 min (p = 0.01) giving the hospital more time to prepare transfusion components. 78% patients received pre-hospital tranexamic acid (TXA; improved from 70%, p = 0.67, ns). Concentrated Red Cell (CRC): Fresh Frozen Plasma (FFP) ratio was always less than 2:1 and below 1.4:1 at 90 min, compared to 2013-15 when CRC: FFP ratios did not drop to below 2:1 until 150 min after arrival in the ED. Mean time for Full Blood Count (FBC; 46 mins versus 81; p = 0.004) and clotting (53 mins versus 119; p<0.001) result was reduced. Survival to hospital discharge was unchanged (66% versus 63%; p = 1.00 ns). CONCLUSIONS: Code Red practice has improved in several ways since our last survey with earlier Code Red activation, more patients receiving pre-hospital transfusion and improved CRC:FFP ratios. Interventions such as earlier on scene Code Red activation, provision of pre-hospital TXA, Emergency Department (ED) resuscitation room pre-thawed FFP and point-of-care viscoelastic coagulation testing have all contributed to these improvements in transfusion practice in Scotland.
Assuntos
Transfusão de Componentes Sanguíneos/estatística & dados numéricos , Transfusão de Sangue/métodos , Hemorragia/terapia , Ressuscitação/métodos , Ferimentos e Lesões/terapia , Adulto , Feminino , Hemorragia/etiologia , Hemorragia/mortalidade , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Plasma , Guias de Prática Clínica como Assunto , Estudos Retrospectivos , Escócia , Taxa de Sobrevida , Centros de Traumatologia , Resultado do Tratamento , Ferimentos e Lesões/mortalidadeRESUMO
Androgen receptor (AR) signalling is a key prostate cancer (PC) driver, even in advanced 'castrate-resistant' disease (CRPC). To systematically identify microRNAs (miRs) modulating AR activity in lethal disease, hormone-responsive and -resistant PC cells expressing a luciferase-based AR reporter were transfected with a miR inhibitor library; 78 inhibitors significantly altered AR activity. Upon validation, miR-346, miR-361-3p and miR-197 inhibitors markedly reduced AR transcriptional activity, mRNA and protein levels, increased apoptosis, reduced proliferation, repressed EMT, and inhibited PC migration and invasion, demonstrating additive effects with AR inhibition. Corresponding miRs increased AR activity through a novel and anti-dogmatic mechanism of direct association with AR 6.9 kb 3'UTR and transcript stabilisation. In addition, miR-346 and miR-361-3p modulation altered levels of constitutively active AR variants, and inhibited variant-driven PC cell proliferation, so may contribute to persistent AR signalling in CRPC in the absence of circulating androgens. Pathway analysis of AGO-PAR-CLIP-identified miR targets revealed roles in DNA replication and repair, cell cycle, signal transduction and immune function. Silencing these targets, including tumour suppressors ARHGDIA and TAGLN2, phenocopied miR effects, demonstrating physiological relevance. MiR-346 additionally upregulated the oncogene, YWHAZ, which correlated with grade, biochemical relapse and metastasis in patients. These AR-modulatory miRs and targets correlated with AR activity in patient biopsies, and were elevated in response to long-term enzalutamide treatment of patient-derived CRPC xenografts. In summary, we identified miRs that modulate AR activity in PC and CRPC, via novel mechanisms, and may represent novel PC therapeutic targets.
Assuntos
MicroRNAs/fisiologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/fisiologia , Regiões 3' não Traduzidas , Elementos Antissenso (Genética) , Benzamidas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/patologia , Transdução de SinaisRESUMO
Aggressive lethal prostate cancer is characterised by tumour invasion, metastasis and androgen resistance. Understanding the mechanisms by which localised disease progresses to advanced lethal stages is key to the development of effective therapies. Here we have identified a novel role for the transcription factor, SOX9, as a driver of aggressive invasive prostate cancer. Using genetically modified mouse models, we show that increased Sox9 expression in the prostate epithelia of animals with Pten loss leads to a highly invasive phenotype and metastasis. In depth analysis of these mice and related in vitro models reveals that SOX9 acts a key regulator of various processes that together promote tumour progression. We show that this factor promotes cell lineage plasticity with cells acquiring properties of basal stem cells and an increase in proliferation. In addition, increased SOX9 leads to changes in cytoskeleton and adhesion, deposition of extracellular matrix and epithelia to mesenchyme transition, properties of highly invasive cells. Analysis of castrated mice showed that the invasive phenotype driven by SOX9 is independent of androgen levels. Our study has identified a novel driver of prostate cancer progression and highlighted the cellular and molecular processes that are regulated by Sox9 to achieve invasive disease.
RESUMO
Purpose: Persistent androgen receptor (AR) signaling drives castration-resistant prostate cancer (CRPC) and confers resistance to AR-targeting therapies. Novel therapeutic strategies to overcome this are urgently required. We evaluated how bromodomain and extra-terminal (BET) protein inhibitors (BETi) abrogate aberrant AR signaling in CRPC.Experimental Design: We determined associations between BET expression, AR-driven transcription, and patient outcome; and the effect and mechanism by which chemical BETi (JQ1 and GSK1210151A; I-BET151) and BET family protein knockdown regulates AR-V7 expression and AR signaling in prostate cancer models.Results: Nuclear BRD4 protein expression increases significantly (P ≤ 0.01) with castration resistance in same patient treatment-naïve (median H-score; interquartile range: 100; 100-170) and CRPC (150; 110-200) biopsies, with higher expression at diagnosis associating with worse outcome (HR, 3.25; 95% CI, 1.50-7.01; P ≤ 0.001). BRD2, BRD3, and BRD4 RNA expression in CRPC biopsies correlates with AR-driven transcription (all P ≤ 0.001). Chemical BETi, and combined BET family protein knockdown, reduce AR-V7 expression and AR signaling. This was not recapitulated by C-MYC knockdown. In addition, we show that BETi regulates RNA processing thereby reducing alternative splicing and AR-V7 expression. Furthermore, BETi reduce growth of prostate cancer cells and patient-derived organoids with known AR mutations, AR amplification and AR-V7 expression. Finally, BETi, unlike enzalutamide, decreases persistent AR signaling and growth (P ≤ 0.001) of a patient-derived xenograft model of CRPC with AR amplification and AR-V7 expression.Conclusions: BETi merit clinical evaluation as inhibitors of AR splicing and function, with trials demonstrating their blockade in proof-of-mechanism pharmacodynamic studies. Clin Cancer Res; 24(13); 3149-62. ©2018 AACR.
Assuntos
Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Processamento Alternativo , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Prognóstico , Neoplasias de Próstata Resistentes à Castração/mortalidade , Neoplasias de Próstata Resistentes à Castração/patologia , Isoformas de Proteínas , RNA Interferente Pequeno/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Resultado do TratamentoRESUMO
A broad range of technologies have been developed to enable three dimensional (3D) cell culture. Few if any however are adaptable for routine everyday use in a straightforward and cost effective manner. Alvetex(®) is a rigid highly porous polystyrene scaffold designed specifically to enable routine 3D cell culture. The scaffold is engineered into thin membranes that fit into conventional cell culture plasticware. The material is inert and offers a polystyrene substrate familiar to cell biologists worldwide. The 3D geometry of the scaffold provides the environment in which cells grow, differentiate, and function to form close relationships with adjacent cells thus creating the equivalent of a thin tissue layer in vitro. This chapter introduces the features required by a technology that enables routine 3D cell culture. Using Alvetex(®) as a product that satisfies these requirements, its application is demonstrated for the growth of a recognised cell line. Procedures detailing the use of Alvetex(®) for 3D cell culture are provided. This is followed by a series of detailed methods describing ways to analyse such cultures including histological techniques, immunocytochemistry, and scanning electron microscopy. Examples of data generated from these methods are shown in the corresponding figures. Additional notes are also included where further information about certain procedures is required. The use of Alvetex(®) in combination with these methods will enable investigators to routinely produce complex 3D cultures to research the growth, differentiation, and function of cells in new ways.