RESUMEN
Non-small cell lung cancers (NSCLCs) harboring KEAP1 mutations are often resistant to immunotherapy. Here, we show that KEAP1 targets EMSY for ubiquitin-mediated degradation to regulate homologous recombination repair (HRR) and anti-tumor immunity. Loss of KEAP1 in NSCLC induces stabilization of EMSY, producing a BRCAness phenotype, i.e., HRR defects and sensitivity to PARP inhibitors. Defective HRR contributes to a high tumor mutational burden that, in turn, is expected to prompt an innate immune response. Notably, EMSY accumulation suppresses the type I interferon response and impairs innate immune signaling, fostering cancer immune evasion. Activation of the type I interferon response in the tumor microenvironment using a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of KEAP1-mutant tumors. Our results suggest that targeting PARP and STING pathways, individually or in combination, represents a therapeutic strategy in NSCLC patients harboring alterations in KEAP1.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/inmunología , Interferón Tipo I/metabolismo , Neoplasias Pulmonares/inmunología , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Reparación del ADN por Recombinación/genética , Proteínas Represoras/metabolismo , Escape del Tumor/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Inmunidad Innata/genética , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Mutación , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Represoras/genética , Transducción de Señal/genética , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.
Asunto(s)
Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Glucosa-6-Fosfato Isomerasa/metabolismo , Glucólisis/genética , Fosforilación Oxidativa , Vía de Pentosa Fosfato/fisiología , Células Th17/metabolismo , Animales , Hipoxia de la Célula/genética , Hipoxia de la Célula/inmunología , Quimera/genética , Cromatografía de Gases , Cromatografía Liquida , Infecciones por Clostridium/inmunología , Citocinas/deficiencia , Citocinas/genética , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Glucólisis/inmunología , Homeostasis/genética , Homeostasis/inmunología , Inflamación/genética , Inflamación/inmunología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiología , Vía de Pentosa Fosfato/genética , Vía de Pentosa Fosfato/inmunología , RNA-Seq , Análisis de la Célula Individual , Células Th17/inmunología , Células Th17/patologíaRESUMEN
Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Neoplasias Pulmonares/patología , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular Tumoral , Movimiento Celular , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Femenino , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Estimación de Kaplan-Meier , Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Metástasis de la Neoplasia , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Activación TranscripcionalRESUMEN
The circadian clock controls metabolic and physiological processes through finely tuned molecular mechanisms. The clock is remarkably plastic and adapts to exogenous "zeitgebers," such as light and nutrition. How a pathological condition in a given tissue influences systemic circadian homeostasis in other tissues remains an unanswered question of conceptual and biomedical importance. Here, we show that lung adenocarcinoma operates as an endogenous reorganizer of circadian metabolism. High-throughput transcriptomics and metabolomics revealed unique signatures of transcripts and metabolites cycling exclusively in livers of tumor-bearing mice. Remarkably, lung cancer has no effect on the core clock but rather reprograms hepatic metabolism through altered pro-inflammatory response via the STAT3-Socs3 pathway. This results in disruption of AKT, AMPK, and SREBP signaling, leading to altered insulin, glucose, and lipid metabolism. Thus, lung adenocarcinoma functions as a potent endogenous circadian organizer (ECO), which rewires the pathophysiological dimension of a distal tissue such as the liver. PAPERCLIP.
Asunto(s)
Adenocarcinoma/fisiopatología , Relojes Circadianos , Hígado/fisiopatología , Neoplasias Pulmonares/fisiopatología , Adenocarcinoma del Pulmón , Animales , Citocinas/genética , Glucosa/metabolismo , Homeostasis , Insulina/metabolismo , Ratones , Transducción de SeñalRESUMEN
Advancements in precision oncology over the past decades have led to new therapeutic interventions, but the efficacy of such treatments is generally limited by an adaptive process that fosters drug resistance1. In addition to genetic mutations2, recent research has identified a role for non-genetic plasticity in transient drug tolerance3 and the acquisition of stable resistance4,5. However, the dynamics of cell-state transitions that occur in the adaptation to cancer therapies remain unknown and require a systems-level longitudinal framework. Here we demonstrate that resistance develops through trajectories of cell-state transitions accompanied by a progressive increase in cell fitness, which we denote as the 'resistance continuum'. This cellular adaptation involves a stepwise assembly of gene expression programmes and epigenetically reinforced cell states underpinned by phenotypic plasticity, adaptation to stress and metabolic reprogramming. Our results support the notion that epithelial-to-mesenchymal transition or stemness programmes-often considered a proxy for phenotypic plasticity-enable adaptation, rather than a full resistance mechanism. Through systematic genetic perturbations, we identify the acquisition of metabolic dependencies, exposing vulnerabilities that can potentially be exploited therapeutically. The concept of the resistance continuum highlights the dynamic nature of cellular adaptation and calls for complementary therapies directed at the mechanisms underlying adaptive cell-state transitions.
Asunto(s)
Adaptación Fisiológica , Plasticidad de la Célula , Resistencia a Antineoplásicos , Neoplasias , Femenino , Humanos , Ratones , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Línea Celular Tumoral , Plasticidad de la Célula/efectos de los fármacos , Plasticidad de la Célula/genética , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , FenotipoRESUMEN
Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs.
Asunto(s)
Carcinogénesis , Modelos Animales de Enfermedad , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Animales , Humanos , Neoplasias Hepáticas/secundario , Metástasis Linfática , Ratones , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Carcinoma Pulmonar de Células Pequeñas/secundarioRESUMEN
Pancreatic ductal adenocarcinoma is a lethal disease characterized by late diagnosis, propensity for early metastasis and resistance to chemotherapy. Little is known about the mechanisms that drive innate therapeutic resistance in pancreatic cancer. The ataxia-telangiectasia group D-associated gene (ATDC) is overexpressed in pancreatic cancer and promotes tumor growth and metastasis. Our study reveals that increased ATDC levels protect cancer cells from reactive oxygen species (ROS) via stabilization of nuclear factor erythroid 2-related factor 2 (NRF2). Mechanistically, ATDC binds to Kelch-like ECH-associated protein 1 (KEAP1), the principal regulator of NRF2 degradation, and thereby prevents degradation of NRF2 resulting in activation of a NRF2-dependent transcriptional program, reduced intracellular ROS and enhanced chemoresistance. Our findings define a novel role of ATDC in regulating redox balance and chemotherapeutic resistance by modulating NRF2 activity.
Asunto(s)
Carcinogénesis/genética , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias Pancreáticas/fisiopatología , Factores de Transcripción/metabolismo , Humanos , Unión Proteica , Neoplasias PancreáticasRESUMEN
Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers1,2, the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Valina-ARNt Ligasa , Valina , Animales , Disponibilidad Biológica , Sistemas CRISPR-Cas , Dieta , Complejo I de Transporte de Electrón/genética , Transportador de Aminoácidos Neutros Grandes 1 , Ratones , Mitocondrias/metabolismo , Fosforilación Oxidativa , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , ARN de Transferencia/genética , Valina/metabolismo , Valina-ARNt Ligasa/metabolismoRESUMEN
Takahashi et al. (2020) conduct a focused CRISPR/Cas9 screen against NRF2 target and other redox regulatory genes in both 2D- and 3D-culture systems, uncovering a vulnerability of spheroid cancer cells deprived of extracellular matrix to undergo ferroptosis.
Asunto(s)
Ferroptosis , Factor 2 Relacionado con NF-E2 , Proliferación Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cultura , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de SeñalRESUMEN
Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbon units from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Glicina Hidroximetiltransferasa/metabolismo , Mitocondrias/genética , Iniciación de la Cadena Peptídica Traduccional , ARN de Transferencia de Metionina/química , Serina/química , Animales , Apoptosis , Neoplasias de la Mama/metabolismo , Sistemas CRISPR-Cas , Proliferación Celular , Citosol/metabolismo , Femenino , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Glicina Hidroximetiltransferasa/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Procesamiento Proteico-Postraduccional , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Serina/genética , Serina/metabolismo , Tetrahidrofolatos/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Kinases are key players in cancer-relevant pathways and are the targets of many successful precision cancer therapies. Phosphoproteomics is a powerful approach to study kinase activity and has been used increasingly for the characterization of tumor samples leading to the identification of novel chemotherapeutic targets and biomarkers. Finding co-regulated phosphorylation sites which represent potential kinase-substrate sets or members of the same signaling pathway allows us to harness these data to identify clinically relevant and targetable alterations in signaling cascades. Unfortunately, studies have found that databases of co-regulated phosphorylation sites are only experimentally supported in a small number of substrate sets. To address the inherent challenge of defining co-regulated phosphorylation modules relevant to a given dataset, we developed PhosphoDisco, a toolkit for determining co-regulated phosphorylation modules. We applied this approach to tandem mass spectrometry based phosphoproteomic data for breast and non-small cell lung cancer and identified canonical as well as putative new phosphorylation site modules. Our analysis identified several interesting modules in each cohort. Among these was a new cell cycle checkpoint module enriched in basal breast cancer samples and a module of PRKC isozymes putatively co-regulated by CDK12 in lung cancer. We demonstrate that modules defined by PhosphoDisco can be used to further personalized cancer treatment strategies by establishing active signaling pathways in a given patient tumor or set of tumors, and in providing new ways to classify tumors based on signaling activity.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Fosforilación , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression and play an important role in many developmental processes. We report here that expression of microRNA-145 (miR-145) is low in self-renewing human embryonic stem cells (hESCs) but highly upregulated during differentiation. We identify the pluripotency factors OCT4, SOX2, and KLF4 as direct targets of miR-145 and show that endogenous miR-145 represses the 3' untranslated regions of OCT4, SOX2, and KLF4. Increased miR-145 expression inhibits hESC self-renewal, represses expression of pluripotency genes, and induces lineage-restricted differentiation. Loss of miR-145 impairs differentiation and elevates OCT4, SOX2, and KLF4. Furthermore, we find that the miR-145 promoter is bound and repressed by OCT4 in hESCs. This work reveals a direct link between the core reprogramming factors and miR-145 and uncovers a double-negative feedback loop involving OCT4, SOX2, KLF4, and miR-145.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , MicroARNs/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/citología , Factores de Transcripción SOXB1/genética , Regiones no Traducidas 3'/metabolismo , Línea Celular , Células Madre Embrionarias/metabolismo , Humanos , Factor 4 Similar a Kruppel , Células Madre Pluripotentes/metabolismoRESUMEN
Environmental nutrient levels impact cancer cell metabolism, resulting in context-dependent gene essentiality. Here, using loss-of-function screening based on RNA interference, we show that environmental oxygen levels are a major driver of differential essentiality between in vitro model systems and in vivo tumours. Above the 3-8% oxygen concentration typical of most tissues, we find that cancer cells depend on high levels of the iron-sulfur cluster biosynthetic enzyme NFS1. Mammary or subcutaneous tumours grow despite suppression of NFS1, whereas metastatic or primary lung tumours do not. Consistent with a role in surviving the high oxygen environment of incipient lung tumours, NFS1 lies in a region of genomic amplification present in lung adenocarcinoma and is most highly expressed in well-differentiated adenocarcinomas. NFS1 activity is particularly important for maintaining the iron-sulfur co-factors present in multiple cell-essential proteins upon exposure to oxygen compared to other forms of oxidative damage. Furthermore, insufficient iron-sulfur cluster maintenance robustly activates the iron-starvation response and, in combination with inhibition of glutathione biosynthesis, triggers ferroptosis, a non-apoptotic form of cell death. Suppression of NFS1 cooperates with inhibition of cysteine transport to trigger ferroptosis in vitro and slow tumour growth. Therefore, lung adenocarcinomas select for expression of a pathway that confers resistance to high oxygen tension and protects cells from undergoing ferroptosis in response to oxidative damage.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Muerte Celular , Proteínas Hierro-Azufre/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Animales , Liasas de Carbono-Azufre/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Muerte Celular/genética , Línea Celular Tumoral , Cisteína/metabolismo , Glutatión/biosíntesis , Humanos , Neoplasias Pulmonares/genética , Ratones , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Estrés Oxidativo/efectos de los fármacos , Oxígeno/metabolismo , Oxígeno/farmacología , Interferencia de ARNRESUMEN
Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr-double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Glutatión Reductasa/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Estrés Oxidativo/fisiología , Tiorredoxina Reductasa 1/metabolismo , Animales , Antioxidantes/metabolismo , Daño del ADN/fisiología , Progresión de la Enfermedad , Regulación de la Expresión Génica/fisiología , Glutatión/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Metaboloma/fisiología , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-ReducciónRESUMEN
Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.
Asunto(s)
Citometría de Flujo , Técnicas Analíticas Microfluídicas , Microfluídica , Neoplasias/diagnóstico , Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Citometría de Flujo/métodos , Perfilación de la Expresión Génica/métodos , Ratones , Microfluídica/métodos , Neoplasias/genética , Células Neoplásicas Circulantes/patología , Análisis de la Célula Individual/métodos , TranscriptomaRESUMEN
Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of cancer. Using a Kras(G12D)-driven lung cancer model, we performed functional characterization of a panel of tumour suppressor genes with known loss-of-function alterations in human lung cancer. Cre-dependent somatic activation of oncogenic Kras(G12D) combined with CRISPR/Cas9-mediated genome editing of tumour suppressor genes resulted in lung adenocarcinomas with distinct histopathological and molecular features. This rapid somatic genome engineering approach enables functional characterization of putative cancer genes in the lung and other tissues using autochthonous mouse models. We anticipate that this approach can be used to systematically dissect the complex catalogue of mutations identified in cancer genome sequencing studies.
Asunto(s)
Adenocarcinoma/genética , Edición Génica/métodos , Neoplasias Pulmonares/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Modelos Animales de Enfermedad , Genes Supresores de Tumor , Genoma , Humanos , Lentivirus/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Mutación/genéticaRESUMEN
The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the ß-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of ß-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.
Asunto(s)
Sistemas CRISPR-Cas , Genes Supresores de Tumor , Ingeniería Genética/métodos , Hígado/metabolismo , Mutagénesis/genética , Mutación/genética , Oncogenes/genética , Animales , Secuencia de Bases , Transformación Celular Neoplásica/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Femenino , Genes p53/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Metabolismo de los Lípidos , Hígado/citología , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Datos de Secuencia Molecular , Fosfohidrolasa PTEN/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , beta Catenina/genéticaRESUMEN
Redox imbalance is defined by disruption in oxidative and reductive pathways and has a central role in cancer initiation, development, and treatment. Although redox imbalance has traditionally been characterized by high levels of oxidative stress, emerging evidence suggests that an overly reductive environment is just as detrimental to cancer proliferation. Reductive stress is defined by heightened levels of antioxidants, including glutathione and elevated NADH, compared with oxidized NAD, which disrupts central biochemical pathways required for proliferation. With the advent of new technologies that measure and manipulate reductive stress, the sensors and drivers of this overlooked metabolic stress are beginning to be revealed. In certain genetically defined cancers, targeting reductive stress pathways may be an effective strategy. Redox-based pathways are gaining recognition as essential 'regulatory hubs,' and a broader understanding of reductive stress signaling promises not only to reveal new insights into metabolic homeostasis but also potentially to transform therapeutic options in cancer.
Asunto(s)
Neoplasias , Estrés Oxidativo , Humanos , Antioxidantes/uso terapéutico , Oxidación-ReducciónRESUMEN
Cellular redox homeostasis is an essential, dynamic process that ensures the balance between reducing and oxidizing reactions within cells and thus has implications across all areas of biology. Changes in levels of reactive oxygen species can disrupt redox homeostasis, leading to oxidative or reductive stress that contributes to the pathogenesis of many malignancies, including cancer. From transformation and tumor initiation to metastatic dissemination, increasing reactive oxygen species in cancer cells can paradoxically promote or suppress the tumorigenic process, depending on the extent of redox stress, its spatiotemporal characteristics and the tumor microenvironment. Here we review how redox regulation influences tumorigenesis, highlighting therapeutic opportunities enabled by redox-related alterations in cancer cells.