RESUMO
The Warburg effect is a hallmark of cancer that refers to the preference of cancer cells to metabolize glucose anaerobically rather than aerobically1,2. This results in substantial accumulation of lacate, the end product of anaerobic glycolysis, in cancer cells3. However, how cancer metabolism affects chemotherapy response and DNA repair in general remains incompletely understood. Here we report that lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair. Lactylation of NBS1 at lysine 388 (K388) is essential for MRE11-RAD50-NBS1 (MRN) complex formation and the accumulation of HR repair proteins at the sites of DNA double-strand breaks. Furthermore, we identify TIP60 as the NBS1 lysine lactyltransferase and the 'writer' of NBS1 K388 lactylation, and HDAC3 as the NBS1 de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcome of neoadjuvant chemotherapy, and lactate reduction using either genetic depletion of lactate dehydrogenase A (LDHA) or stiripentol, a lactate dehydrogenase A inhibitor used clinically for anti-epileptic treatment, inhibited NBS1 K388 lactylation, decreased DNA repair efficacy and overcame resistance to chemotherapy. In summary, our work identifies NBS1 lactylation as a critical mechanism for genome stability that contributes to chemotherapy resistance and identifies inhibition of lactate production as a promising therapeutic cancer strategy.
Assuntos
Proteínas de Ciclo Celular , Resistencia a Medicamentos Antineoplásicos , Ácido Láctico , Proteínas Nucleares , Reparo de DNA por Recombinação , Animais , Feminino , Humanos , Masculino , Camundongos , Hidrolases Anidrido Ácido/metabolismo , Anaerobiose , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Instabilidade Genômica , Ácido Láctico/metabolismo , Lisina/química , Lisina/metabolismo , Lisina Acetiltransferase 5/metabolismo , Lisina Acetiltransferase 5/genética , Proteína Homóloga a MRE11/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Organoides , Glicólise , Terapia Neoadjuvante , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/deficiência , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Anticonvulsivantes/farmacologiaRESUMO
B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.
Assuntos
Retrovirus Endógenos , Imunoterapia , Neoplasias Pulmonares , Animais , Humanos , Camundongos , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/terapia , Adenocarcinoma de Pulmão/virologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Carcinoma Pulmonar de Células não Pequenas/virologia , Modelos Animais de Doenças , Retrovirus Endógenos/imunologia , Imunoterapia/métodos , Pulmão/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/virologia , Microambiente Tumoral , Linfócitos B/imunologia , Estudos de Coortes , Anticorpos/imunologia , Anticorpos/uso terapêuticoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) shows pronounced epithelial and mesenchymal cancer cell populations1-4. Cellular heterogeneity in PDAC is an important feature in disease subtype specification3-5, but how distinct PDAC subpopulations interact, and the molecular mechanisms that underlie PDAC cell fate decisions, are incompletely understood. Here we identify the BMP inhibitor GREM16,7 as a key regulator of cellular heterogeneity in pancreatic cancer in human and mouse. Grem1 inactivation in established PDAC in mice resulted in a direct conversion of epithelial into mesenchymal PDAC cells within days, suggesting that persistent GREM1 activity is required to maintain the epithelial PDAC subpopulations. By contrast, Grem1 overexpression caused an almost complete 'epithelialization' of highly mesenchymal PDAC, indicating that high GREM1 activity is sufficient to revert the mesenchymal fate of PDAC cells. Mechanistically, Grem1 was highly expressed in mesenchymal PDAC cells and inhibited the expression of the epithelial-mesenchymal transition transcription factors Snai1 (also known as Snail) and Snai2 (also known as Slug) in the epithelial cell compartment, therefore restricting epithelial-mesenchymal plasticity. Thus, constant suppression of BMP activity is essential to maintain epithelial PDAC cells, indicating that the maintenance of the cellular heterogeneity of pancreatic cancer requires continuous paracrine signalling elicited by a single soluble factor.
Assuntos
Transição Epitelial-Mesenquimal , Peptídeos e Proteínas de Sinalização Intercelular , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/patologia , Camundongos , Neoplasias Pancreáticas/patologia , Fatores de Transcrição da Família SnailRESUMO
Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fator de Transcrição GATA2/metabolismo , Redes Reguladoras de Genes , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ras/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Fator de Transcrição GATA2/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Proteínas ras/genéticaRESUMO
Tubular epithelia are a basic building block of organs and a common site of cancer occurrence1-4. During tumorigenesis, transformed cells overproliferate and epithelial architecture is disrupted. However, the biophysical parameters that underlie the adoption of abnormal tumour tissue shapes are unknown. Here we show in the pancreas of mice that the morphology of epithelial tumours is determined by the interplay of cytoskeletal changes in transformed cells and the existing tubular geometry. To analyse the morphological changes in tissue architecture during the initiation of cancer, we developed a three-dimensional whole-organ imaging technique that enables tissue analysis at single-cell resolution. Oncogenic transformation of pancreatic ducts led to two types of neoplastic growth: exophytic lesions that expanded outwards from the duct and endophytic lesions that grew inwards to the ductal lumen. Myosin activity was higher apically than basally in wild-type cells, but upon transformation this gradient was lost in both lesion types. Three-dimensional vertex model simulations and a continuum theory of epithelial mechanics, which incorporate the cytoskeletal changes observed in transformed cells, indicated that the diameter of the source epithelium instructs the morphology of growing tumours. Three-dimensional imaging revealed that-consistent with theory predictions-small pancreatic ducts produced exophytic growth, whereas large ducts deformed endophytically. Similar patterns of lesion growth were observed in tubular epithelia of the liver and lung; this finding identifies tension imbalance and tissue curvature as fundamental determinants of epithelial tumorigenesis.
Assuntos
Fenômenos Biomecânicos , Polaridade Celular , Transformação Celular Neoplásica , Morfogênese , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Animais , Humanos , Camundongos , Organoides/patologia , Estresse MecânicoRESUMO
The regeneration-associated gene (RAG) expression program is activated in injured peripheral neurons after axotomy and enables long-distance axon re-growth. Over 1000 genes are regulated, and many transcription factors are upregulated or activated as part of this response. However, a detailed picture of how RAG expression is regulated is lacking. In particular, the transcriptional targets and specific functions of the various transcription factors are unclear. Jun was the first-regeneration-associated transcription factor identified and the first shown to be functionally important. Here we fully define the role of Jun in the RAG expression program in regenerating facial motor neurons. At 1, 4 and 14 days after axotomy, Jun upregulates 11, 23 and 44% of the RAG program, respectively. Jun functions relevant to regeneration include cytoskeleton production, metabolic functions and cell activation, and the downregulation of neurotransmission machinery. In silico analysis of promoter regions of Jun targets identifies stronger over-representation of AP1-like sites than CRE-like sites, although CRE sites were also over-represented in regions flanking AP1 sites. Strikingly, in motor neurons lacking Jun, an alternative SRF-dependent gene expression program is initiated after axotomy. The promoters of these newly expressed genes exhibit over-representation of CRE sites in regions near to SRF target sites. This alternative gene expression program includes plasticity-associated transcription factors and leads to an aberrant early increase in synapse density on motor neurons. Jun thus has the important function in the early phase after axotomy of pushing the injured neuron away from a plasticity response and towards a regenerative phenotype.
Assuntos
Axônios , Regeneração Nervosa , Axônios/metabolismo , Axotomia , Neurônios Motores/metabolismo , Regeneração Nervosa/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology due to improvements in capacity and accuracy of microscopy techniques. Here, we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary gland organoids imaged over 24 h with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin that implements active mesh deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction.
Assuntos
Núcleo Celular , Redes Neurais de Computação , Animais , Camundongos , Microscopia de Fluorescência/métodos , Processamento de Imagem Assistida por Computador/métodosRESUMO
Oxaliplatin is the first-line regime for advanced gastric cancer treatment, while its resistance is a major problem that leads to the failure of clinical treatments. Tumor cell heterogeneity has been considered as one of the main causes for drug resistance in cancer. In this study, the mechanism of oxaliplatin resistance was investigated through in vitro human gastric cancer organoids and gastric cancer oxaliplatin-resistant cell lines and in vivo subcutaneous tumorigenicity experiments. The in vitro and in vivo results indicated that CD133+ stem cell-like cells are the main subpopulation and PARP1 is the central gene mediating oxaliplatin resistance in gastric cancer. It was found that PARP1 can effectively repair DNA damage caused by oxaliplatin by means of mediating the opening of base excision repair pathway, leading to the occurrence of drug resistance. The CD133+ stem cells also exhibited upregulated expression of N6-methyladenosine (m6A) mRNA and its writer METTL3 as showed by immunoprecipitation followed by sequencing and transcriptome analysis. METTTL3 enhances the stability of PARP1 by recruiting YTHDF1 to target the 3'-untranslated Region (3'-UTR) of PARP1 mRNA. The CD133+ tumor stem cells can regulate the stability and expression of m6A to PARP1 through METTL3, and thus exerting the PARP1-mediated DNA damage repair ability. Therefore, our study demonstrated that m6A Methyltransferase METTL3 facilitates oxaliplatin resistance in CD133+ gastric cancer stem cells by Promoting PARP1 mRNA stability which increases base excision repair pathway activity.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Metiltransferases/metabolismo , Células-Tronco Neoplásicas/patologia , Oxaliplatina/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Estabilidade de RNA , Neoplasias Gástricas/tratamento farmacológico , Antígeno AC133 , Animais , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Criança , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Prognóstico , RNA Mensageiro , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
WDR62 is the second most common genetic alteration associated with microcephaly. It has been shown that Wdr62 is required for germ cell meiosis initiation in mice, and the majority of male germ cells are lost in the meiotic defect of first wave spermatogenesis in Wdr62 mutants. Strikingly, in this study, we found that the initiation of meiosis following spermatogenesis was not affected and the germ cells were gradually repopulated at later developmental stages. However, most germ cells were arrested at metaphase of meiosis I and no mature sperm were detected in epididymides. Further, this study demonstrated that metaphase I arrest of Wdr62-deficient spermatocytes was caused by asymmetric distribution of the centrosome and aberrant spindle assembly. Also, mechanistic studies demonstrated that WDR62 interacts with centrosome-associated protein CEP170, and deletion of Wdr62 causes downregulation of the CEP170 protein, which in turn leads to the aberrant spindle assembly. In summary, this study indicates that the meiosis of first wave spermatogenesis and the following spermatogenesis started from spermatogonium is probably regulated by different mechanisms. We also demonstrated a new function of WDR62 in germ cell meiosis, through its interaction with CEP170.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fuso Acromático/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Centrossomo/metabolismo , Masculino , Meiose/genética , Meiose/fisiologia , Metáfase/genética , Metáfase/fisiologia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1007463.].
RESUMO
Mutations of WD repeat domain 62 (WDR62) lead to autosomal recessive primary microcephaly (MCPH), and down-regulation of WDR62 expression causes the loss of neural progenitor cells (NPCs). However, how WDR62 is regulated and hence controls neurogenesis and brain size remains elusive. Here, we demonstrate that mitogen-activated protein kinase kinase kinase 3 (MEKK3) forms a complex with WDR62 to promote c-Jun N-terminal kinase (JNK) signaling synergistically in the control of neurogenesis. The deletion of Mekk3, Wdr62, or Jnk1 resulted in phenocopied defects, including premature NPC differentiation. We further showed that WDR62 protein is positively regulated by MEKK3 and JNK1 in the developing brain and that the defects of wdr62 deficiency can be rescued by the transgenic expression of JNK1. Meanwhile, WDR62 is also negatively regulated by T1053 phosphorylation, leading to the recruitment of F-box and WD repeat domain-containing protein 7 (FBW7) and proteasomal degradation. Our findings demonstrate that the coordinated reciprocal and bidirectional regulation among MEKK3, FBW7, WDR62, and JNK1, is required for fine-tuned JNK signaling for the control of balanced NPC self-renewal and differentiation during cortical development.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína 7 com Repetições F-Box-WD/fisiologia , MAP Quinase Quinase Quinase 3/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Diferenciação Celular , Proteína 7 com Repetições F-Box-WD/genética , Feminino , Células HEK293 , Humanos , MAP Quinase Quinase Quinase 3/genética , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microcefalia/genética , Microcefalia/fisiopatologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Fosforilação , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Meiosis is a germ cell-specific division that is indispensable for the generation of haploid gametes. However, the regulatory mechanisms of meiotic initiation remain elusive. Here, we report that the Wdr62 (WD40-repeat protein 62) is involved in meiotic initiation as a permissive factor rather than an instructive factor. Knock-out of this gene in a mouse model resulted in female meiotic initiation defects. Further studies demonstrated that Wdr62 is required for RA-induced Stra8 expression via the activation of JNK signaling, and the defects in meiotic initiation from Wdr62-deficient female mice could be partially rescued by JNK1 overexpression in germ cells. More importantly, two novel mutations of the WDR62 gene were detected in patients with premature ovarian insufficiency (POI), and these mutations played dominant-negative roles in regulating Stra8 expression. Hence, this study revealed that Wdr62 is involved in female meiotic initiation via activating JNK signaling, which displays a novel mechanism for regulating meiotic initiation, and mutation of WDR62 is one of the potential etiologies of POI in humans.
Assuntos
Proteínas de Ciclo Celular/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Insuficiência Ovariana Primária/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Povo Asiático/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Células Germinativas , Haploidia , Humanos , Masculino , Meiose , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Insuficiência Ovariana Primária/diagnóstico , Análise de Sequência de DNA , Sequenciamento do ExomaRESUMO
The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Homeostase , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Pele/metabolismo , Animais , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Dasatinibe/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Neoplasias de Células Escamosas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Cicatrização/efeitos dos fármacos , Proteínas de Sinalização YAP , Quinases da Família src/metabolismoRESUMO
Cancer chromosomal instability (CIN) results in an increased rate of change of chromosome number and structure and generates intratumour heterogeneity. CIN is observed in most solid tumours and is associated with both poor prognosis and drug resistance. Understanding a mechanistic basis for CIN is therefore paramount. Here we find evidence for impaired replication fork progression and increased DNA replication stress in CIN(+) colorectal cancer (CRC) cells relative to CIN(-) CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three new CIN-suppressor genes (PIGN (also known as MCD4), MEX3C (RKHD2) and ZNF516 (KIAA0222)) encoded on chromosome 18q that are subject to frequent copy number loss in CIN(+) CRC. Chromosome 18q loss was temporally associated with aneuploidy onset at the adenoma-carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage, reduces the frequency of chromosome segregation errors after CIN-suppressor gene silencing, and attenuates segregation errors and DNA damage in CIN(+) cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity.
Assuntos
Instabilidade Cromossômica/genética , Neoplasias Colorretais/genética , Replicação do DNA/genética , Aneuploidia , Linhagem Celular Tumoral , Instabilidade Cromossômica/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/genética , Cromossomos Humanos Par 18/efeitos dos fármacos , Cromossomos Humanos Par 18/genética , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Replicação do DNA/efeitos dos fármacos , Deleção de Genes , Inativação Gênica , Genes Supressores de Tumor , Humanos , Mitose/efeitos dos fármacos , Nucleosídeos/farmacologia , Fosfotransferases/genética , Proteínas de Ligação a RNA/genéticaRESUMO
The ataxia telangiectasia mutated (ATM)-interacting protein ATMIN mediates noncanonical ATM signaling in response to oxidative and replicative stress conditions. Like ATM, ATMIN can function as a tumor suppressor in the hematopoietic system: deletion of Atmin under the control of CD19-Cre results in B-cell lymphomas in aging mice. ATM signaling is essential for lymphopoiesis and hematopoietic stem cell (HSC) function; however, little is known about the role of ATMIN in hematopoiesis. We thus sought to investigate whether the absence of ATMIN would affect primitive hematopoietic cells in an ATM-dependent or -independent manner. Apart from its role in B-cell development, we show that ATMIN has an ATM-independent function in the common myeloid progenitors (CMPs) by deletion of Atmin in the entire hematopoietic system using Vav-Cre. Despite the lack of lymphoma formation, ATMIN-deficient mice developed chronic leukopenia as a result of high levels of apoptosis in B cells and CMPs and induced a compensatory mechanism in which HSCs displayed enhanced cycling. Consequently, ATMIN-deficient HSCs showed impaired regeneration ability with the induction of the DNA oxidative stress response, especially when aged. ATMIN, therefore, has multiple roles in different cell types, and its absence results in perturbed hematopoiesis, especially during stress conditions and aging.
Assuntos
Envelhecimento , Apoptose/genética , Hematopoese/genética , Células-Tronco Hematopoéticas , Estresse Oxidativo/genética , Fatores de Transcrição , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Doença Crônica , Deleção de Genes , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Leucopenia/genética , Leucopenia/metabolismo , Leucopenia/patologia , Camundongos , Camundongos Knockout , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. In vivo this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Reparo do DNA , Ativação Linfocitária/fisiologia , Proteínas Nucleares/fisiologia , Linfócitos T/imunologia , Fatores de Transcrição/fisiologia , Animais , Colite/imunologia , Dano ao DNA , Proteínas de Ligação a DNA , Imunofenotipagem , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Recombinação Genética , Baço/citologia , Baço/metabolismoRESUMO
Fbw7 is well characterised as a stem cell regulator and tumour suppressor, powerfully positioned to control proliferation, differentiation and apoptosis by targeting key transcription factors for ubiquitination and destruction. Evidence in support of these roles continues to accumulate from in vitro studies, mouse models and human patient data. Here we summarise the latest of these findings, highlighting the tumour-suppressive role of Fbw7 in multiple tissues, and the rare circumstances where Fbw7 activity can be oncogenic. We discuss mechanisms that regulate ubiquitination by Fbw7, including ubiquitin-specific proteases such as USP28 that counteract Fbw7 activity and thereby stabilise oncoproteins. Deubiquitination of key Fbw7 substrates to prevent their destruction is beginning to be appreciated as an important pro-tumourigenic mechanism. As the ubiquitin-proteasome system represents a largely untapped field for drug development, the interplay between Fbw7 and its counterpart deubiquitinating enzymes in tumours is likely to attract increasing interest and influence future treatment strategies.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Células-Tronco/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Proteínas de Ciclo Celular/antagonistas & inibidores , Diferenciação Celular/genética , Proliferação de Células , Proteínas F-Box/antagonistas & inibidores , Proteína 7 com Repetições F-Box-WD , Humanos , Neoplasias/patologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transdução de Sinais , Células-Tronco/citologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , UbiquitinaçãoRESUMO
c-Jun, the major component of the AP-1 transcription factor complex, has important functions in cellular proliferation and oncogenic transformation. The RING domain-containing protein RACO-1 functions as a c-Jun coactivator that molecularly links growth factor signalling to AP-1 transactivation. Here we demonstrate that RACO-1 is present as a nuclear dimer and that c-Jun specifically interacts with dimeric RACO-1. Moreover, RACO-1 is identified as a substrate of the arginine methyltransferase PRMT1, which methylates RACO-1 on two arginine residues. Arginine methylation of RACO-1 promotes a conformational change that stabilises RACO-1 by facilitating K63-linked ubiquitin chain formation, and enables RACO-1 dimerisation and c-Jun interaction. Abrogation of PRMT1 function impairs AP-1 activity and results in decreased expression of a large percentage of c-Jun target genes. These results demonstrate that arginine methylation of RACO-1 is required for efficient transcriptional activation by c-Jun/AP-1 and thus identify PRMT1 as an important regulator of c-Jun/AP-1 function.