RESUMEN
Oncogenic RAS-induced senescence (OIS) is an autonomous tumour suppressor mechanism associated with premalignancy1,2. Achieving this phenotype typically requires a high level of oncogenic stress, yet the phenotype provoked by lower oncogenic dosage remains unclear. Here we develop oncogenic RAS dose-escalation models in vitro and in vivo, revealing a RAS dose-driven non-linear continuum of downstream phenotypes. In a hepatocyte OIS model in vivo, ectopic expression of NRAS(G12V) does not induce tumours, in part owing to OIS-driven immune clearance3. Single-cell RNA sequencing analyses reveal distinct hepatocyte clusters with typical OIS or progenitor-like features, corresponding to high and intermediate levels of NRAS(G12V), respectively. When titred down, NRAS(G12V)-expressing hepatocytes become immune resistant and develop tumours. Time-series monitoring at single-cell resolution identifies two distinct tumour types: early-onset aggressive undifferentiated and late-onset differentiated hepatocellular carcinoma. The molecular signature of each mouse tumour type is associated with different progenitor features and enriched in distinct human hepatocellular carcinoma subclasses. Our results define the oncogenic dosage-driven OIS spectrum, reconciling the senescence and tumour initiation phenotypes in early tumorigenesis.
RESUMEN
Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art λFLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.
Asunto(s)
Aumento de la Imagen/instrumentación , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Confocal/instrumentación , Imagen Molecular/instrumentación , Imagen Óptica/instrumentación , Diseño Asistido por Computadora , Diseño de Equipo , Análisis de Falla de Equipo , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/instrumentación , Lentes , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/instrumentaciónRESUMEN
Autophagy is an evolutionarily conserved cellular process in which macromolecules undergo lysosomal degradation. It fulfills essential roles in quality controlling cellular constituents and in energy homeostasis. Basal autophagy is also widely accepted to provide a protective role in aging and aging-related disorders, and its decline with age might precipitate the onset of a variety of diseases. In this review, we discuss the role of basal autophagy in maintaining homeostasis, in part through the maintenance of stem cell populations and the prevention of cellular senescence. We also consider how stress-induced senescence, for example, during oncogene activation and in premalignant disease, might rely on autophagy, and the possibility that the age-associated decline in autophagy might promote tumour development through a variety of mechanisms. Ultimately, evidence suggests that autophagy is required for malignant cancer progression in a number of settings. Thus, autophagy appears to be tumour-suppressive during the early stages of tumorigenesis and tumour-promoting at later stages.
Asunto(s)
Autofagia , Neoplasias , Envejecimiento/metabolismo , Autofagia/fisiología , Senescencia Celular , Humanos , Lisosomas/metabolismo , Neoplasias/patologíaRESUMEN
Both senescence and autophagy have been strongly linked to aging and also cancer development. Numerous molecular, cellular, and physiological changes are known to correlate with an increasing age, yet our understanding of what underlies these changes or how they combine to give rise to the various pathologies associated with aging is still unclear. Levels of autophagy activity are known to decrease with advancing age, in a variety of organisms including mammals. Whereas senescent cells are known to accumulate in our bodies with age. Herein we review evidence from some elegant genetic mouse models linking senescence and also autophagy to aging and cancer. It is especially interesting to note the convergence in the pathological phenotypes of these two processes, senescence and autophagy, in these mouse models.
Asunto(s)
Envejecimiento/patología , Autofagia/fisiología , Senescencia Celular/fisiología , Envejecimiento/fisiología , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Reduced autophagy has been implicated in aging, yet whether its loss can promote aging phenotypes and pathologies in mammals, and how reversible this process is, has never been fully explored. Using inducible short hairpin RNA (shRNA) mouse models, we have recently shown that autophagy inhibition accelerates aging, and that even a temporary block in autophagy can create irreversible damage that increases a cancer risk.
RESUMEN
Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially.
Asunto(s)
Envejecimiento/fisiología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Longevidad/fisiología , Neoplasias , Envejecimiento/genética , Animales , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/genética , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Femenino , Inflamación , Longevidad/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculos , Fenotipo , Proteína Sequestosoma-1/metabolismo , Piel/patologíaRESUMEN
Interleukin-1 alpha (IL-1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL-1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL-1α in these contexts is unknown. We show IL-1α is activated by caspase-5 or caspase-11 cleavage at a conserved site. Caspase-5 drives cleaved IL-1α release after human macrophage inflammasome activation, while IL-1α secretion from murine macrophages only requires caspase-11, with IL-1ß release needing caspase-11 and caspase-1. Importantly, senescent human cells require caspase-5 for the IL-1α-dependent senescence-associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase-11 for the SASP-driven immune surveillance of senescent cells in vivo. Together, we identify IL-1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL-1α is activated during senescence. Thus, targeting caspase-5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.
Asunto(s)
Caspasas/metabolismo , Senescencia Celular , Inflamasomas/metabolismo , Inflamación/metabolismo , Interleucina-1alfa/metabolismo , Animales , Células Cultivadas , Femenino , Células HeLa , Humanos , Interleucina-1alfa/análisis , Ratones , Ratones Endogámicos C57BLRESUMEN
Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for in vivo studies. Here, we have developed an inducible shRNA mouse model targeting Atg5, allowing us to dynamically inhibit autophagy in vivo, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies.
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Proteína 5 Relacionada con la Autofagia/metabolismo , Autofagia , ARN Interferente Pequeño/metabolismo , Animales , Animales Recién Nacidos , Proteína 5 Relacionada con la Autofagia/genética , Regulación hacia Abajo/genética , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Modelos Animales , Fenotipo , Factores de TiempoRESUMEN
Chromosomal instability is a hallmark of human cancer cells, but its role in carcinogenesis remains poorly resolved. Insights into this role have emerged from studies on the tumour suppressor BRCA2, whose inactivation in human cancers causes chromosomal instability through the loss of essential functions of the BRCA2 protein in the normal mechanisms responsible for the replication, repair and segregation of DNA during cell division. Humans who carry heterozygous germline mutations in the BRCA2 gene are highly predisposed to cancers of the breast, ovary, pancreas, prostate and other tissues. Here, we review recent studies that describe genetically engineered mouse models (GEMMs) for pancreatic cancer associated with BRCA2 mutations. These studies not only surprisingly show that BRCA2 does not follow the classical Knudson "two hit" paradigm for tumour suppression, but also highlight features of the interplay between TP53 inactivation and carcinogenesis in the context of BRCA2 deficiency. Thus, the models reveal novel aspects of cancer evolution in carriers of germline BRCA2 mutations, provide new insights into the tumour suppressive role of BRCA2, and establish valuable new preclinical settings for testing approaches to pancreatic cancer therapy; together, these features emphasize the value of GEMMs in cancer research.
Asunto(s)
Proteína BRCA2/metabolismo , Inestabilidad Cromosómica , Cromosomas Humanos/metabolismo , Mutación de Línea Germinal , Neoplasias Experimentales/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Proteína BRCA2/genética , Transformación Celular Neoplásica , Cromosomas Humanos/genética , Reparación del ADN , Replicación del ADN/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Humanos , Ratones , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologíaRESUMEN
Genomic instability is a hallmark of cancer cells, and arises from the aberrations that these cells exhibit in the normal biological mechanisms that repair and replicate the genome, or ensure its accurate segregation during cell division. Increasingly detailed descriptions of cancer genomes have begun to emerge from next-generation sequencing (NGS), providing snapshots of their nature and heterogeneity in different cancers at different stages in their evolution. Here, we attempt to extract from these sequencing studies insights into the role of genome instability mechanisms in carcinogenesis, and to identify challenges impeding further progress.
Asunto(s)
Genoma , Inestabilidad Genómica , Neoplasias/genética , Animales , Cromosomas , Evolución Molecular , Humanos , Modelos GenéticosRESUMEN
Inherited heterozygous BRCA2 mutations predispose carriers to tissue-specific cancers, but somatic deletion of the wild-type allele is considered essential for carcinogenesis. We find in a murine model of familial pancreatic cancer that germline heterozygosity for a pathogenic Brca2 truncation suffices to promote pancreatic ductal adenocarcinomas (PDACs) driven by Kras(G12D), irrespective of Trp53 status. Unexpectedly, tumor cells retain a functional Brca2 allele. Correspondingly, three out of four PDACs from patients inheriting BRCA2(999del5) did not exhibit loss-of-heterozygosity (LOH). Three tumors from these patients displaying LOH were acinar carcinomas, which also developed only in mice with biallelic Brca2 inactivation. We suggest a revised model for tumor suppression by BRCA2 with implications for the therapeutic strategy targeting BRCA2 mutant cancer cells.