RESUMO
NOTCH1 mutant clones occupy the majority of normal human esophagus by middle age but are comparatively rare in esophageal cancers, suggesting NOTCH1 mutations drive clonal expansion but impede carcinogenesis. Here we test this hypothesis. Sequencing NOTCH1 mutant clones in aging human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygous Notch1 mutation confers a competitive advantage over wild-type cells, an effect enhanced by loss of the second allele. Widespread Notch1 loss alters transcription but has minimal effects on the epithelial structure and cell dynamics. In a carcinogenesis model, Notch1 mutations were less prevalent in tumors than normal epithelium. Deletion of Notch1 reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. Notch1 null tumors showed reduced proliferation. We conclude that Notch1 mutations in normal epithelium are beneficial as wild-type Notch1 favors tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.
Assuntos
Neoplasias Esofágicas , Animais , Humanos , Camundongos , Pessoa de Meia-Idade , Carcinogênese/patologia , Epitélio/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Mutação , Receptor Notch1/genéticaRESUMO
Aging normal human oesophagus accumulates TP53 mutant clones. These are the origin of most oesophageal squamous carcinomas, in which biallelic TP53 disruption is almost universal. However, how p53 mutant clones expand and contribute to cancer development is unclear. Here we show that inducing the p53R245W mutant in single oesophageal progenitor cells in transgenic mice confers a proliferative advantage and clonal expansion but does not disrupt normal epithelial structure. Loss of the remaining p53 allele in mutant cells results in genomically unstable p53R245W/null epithelium with giant polyaneuploid cells and copy number altered clones. In carcinogenesis, p53 mutation does not initiate tumour formation, but tumours developing from areas with p53 mutation and LOH are larger and show extensive chromosomal instability compared to lesions arising in wild type epithelium. We conclude that p53 has distinct functions at different stages of carcinogenesis and that LOH within p53 mutant clones in normal epithelium is a critical step in malignant transformation.
Assuntos
Carcinogênese , Proteína Supressora de Tumor p53 , Humanos , Camundongos , Animais , Proteína Supressora de Tumor p53/genética , Carcinogênese/genética , Células Clonais , Esôfago , Camundongos Transgênicos , Instabilidade Cromossômica , MutaçãoRESUMO
In recent years, progress in the study of the lateral organization of the plasma membrane has led to the proposal that mammalian cells use two different organelles to store lipids: intracellular lipid droplets (LDs) and plasma membrane caveolae. Experimental evidence suggests that caveolin (CAV) may act as a sensitive lipid-organizing molecule that physically connects these two lipid-storing organelles. Here, we determine the sequences necessary for efficient sorting of CAV to LDs. We show that targeting is a process cooperatively mediated by two motifs. CAV's central hydrophobic domain (Hyd) anchors CAV to the endoplasmic reticulum (ER). Next, positively charged sequences (Pos-Seqs) mediate sorting of CAVs into LDs. Our findings were confirmed by identifying an equivalent, non-conserved but functionally interchangeable Pos-Seq in ALDI, a bona fide LD-resident protein. Using this information, we were able to retarget a cytosolic protein and convert it to an LD-resident protein. Further studies suggest three requirements for targeting via this mechanism: the positive charge of the Pos-Seq, physical proximity between Pos-Seq and Hyd and a precise spatial orientation between both motifs. The study uncovers remarkable similarities with the signals that target proteins to the membrane of mitochondria and peroxisomes.
Assuntos
Lipídeos/química , Proteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Dados de Sequência MolecularRESUMO
The molecular components of membrane rafts are frequently defined by their biochemical partitioning into detergent-resistant membranes. In the present study, we used a combination of epifluorescence and two-photon microscopy to visualize and quantify whether this insolubility in detergent reflects a pre-existing organization of the PM (plasma membrane). We found that the treatment of cells with cold TX (Triton X-100) promotes a profound remodelling of the PM, including a rapid rearrangement of the glycosphingolipid GM1 and cholesterol into newly formed structures, only partial solubilization of fluid domains and the formation of condensed domains that cover 51% of the remaining membrane. TX does not appear to induce the coalescence of pre-existing domains; instead, the domains that remain after TX treatment seem to be newly formed with a higher degree of condensation than those observed in native membranes. However, when cholesterol was complexed physically by treatment with a second detergent, such as saponin, cholesterol did not separate into the newly formed structures, condensation of the domains was unaltered, and the relative area corresponding to ordered domains increased to occupy 62% of the remaining membrane. Our results suggest that detergent can be used to enrich ordered domains for biochemical analysis, but that TX treatment alone substantially alters the lateral organization of the PM.
Assuntos
Membrana Celular/efeitos dos fármacos , Colesterol/metabolismo , Octoxinol/farmacologia , Animais , Células COS , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Detergentes/farmacologia , Glicoesfingolipídeos/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência por Excitação MultifotônicaRESUMO
In adult skin epidermis and the epithelium lining the esophagus cells are constantly shed from the tissue surface and replaced by cell division. Tracking genetically labelled cells in transgenic mice has given insight into cell behavior, but conflicting models appear consistent with the results. Here, we use an additional transgenic assay to follow cell division in mouse esophagus and the epidermis at multiple body sites. We find that proliferating cells divide at a similar rate, and place bounds on the distribution cell cycle times. By including these results in a common analytic approach, we show that data from eight lineage tracing experiments is consistent with tissue maintenance by a single population of proliferating cells. The outcome of a given cell division is unpredictable but, on average, the likelihood of producing proliferating and differentiating cells is equal, ensuring cellular homeostasis. These findings are key to understanding squamous epithelial homeostasis and carcinogenesis.
Assuntos
Epiderme/crescimento & desenvolvimento , Esôfago/citologia , Células-Tronco/citologia , Animais , Ciclo Celular , Divisão Celular , Proliferação de Células , Esôfago/crescimento & desenvolvimento , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
During aging, progenitor cells acquire mutations, which may generate clones that colonize the surrounding tissue. By middle age, normal human tissues, including the esophageal epithelium (EE), become a patchwork of mutant clones. Despite their relevance for understanding aging and cancer, the processes that underpin mutational selection in normal tissues remain poorly understood. Here, we investigated this issue in the esophageal epithelium of mutagen-treated mice. Deep sequencing identified numerous mutant clones with multiple genes under positive selection, including Notch1, Notch2 and Trp53, which are also selected in human esophageal epithelium. Transgenic lineage tracing revealed strong clonal competition that evolved over time. Clone dynamics were consistent with a simple model in which the proliferative advantage conferred by positively selected mutations depends on the nature of the neighboring cells. When clones with similar competitive fitness collide, mutant cell fate reverts towards homeostasis, a constraint that explains how selection operates in normal-appearing epithelium.
Assuntos
Esôfago/citologia , Mutação , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Linhagem da Célula , Dietilnitrosamina/toxicidade , Epitélio/efeitos dos fármacos , Epitélio/patologia , Epitélio/fisiologia , Esôfago/fisiologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor Notch1/genética , Receptor Notch2/genética , Reprodutibilidade dos Testes , Proteína Supressora de Tumor p53/genéticaRESUMO
As humans age, normal tissues, such as the esophageal epithelium, become a patchwork of mutant clones. Some mutations are under positive selection, conferring a competitive advantage over wild-type cells. We speculated that altering the selective pressure on mutant cell populations may cause them to expand or contract. We tested this hypothesis by examining the effect of oxidative stress from low-dose ionizing radiation (LDIR) on wild-type and p53 mutant cells in the transgenic mouse esophagus. We found that LDIR drives wild-type cells to stop proliferating and differentiate. p53 mutant cells are insensitive to LDIR and outcompete wild-type cells following exposure. Remarkably, combining antioxidant treatment and LDIR reverses this effect, promoting wild-type cell proliferation and p53 mutant differentiation, reducing the p53 mutant population. Thus, p53-mutant cells can be depleted from the normal esophagus by redox manipulation, showing that external interventions may be used to alter the mutational landscape of an aging tissue.
Assuntos
Envelhecimento/fisiologia , Células Epiteliais/fisiologia , Esôfago/fisiologia , Receptores de Estrogênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Antioxidantes , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Estresse Oxidativo , Radiação Ionizante , Receptores de Estrogênio/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Lipid droplets (LDs) are intracellular organelles that provide fatty acids (FAs) to cellular processes including synthesis of membranes and production of metabolic energy. While known to move bidirectionally along microtubules (MTs), the role of LD motion and whether it facilitates interaction with other organelles are unclear. Here we show that during nutrient starvation, LDs and mitochondria relocate on detyrosinated MT from the cell centre to adopt a dispersed distribution. In the cell periphery, LD-mitochondria interactions increase and LDs efficiently supply FAs for mitochondrial beta-oxidation. This cellular adaptation requires the activation of the energy sensor AMPK, which in response to starvation simultaneously increases LD motion, reorganizes the network of detyrosinated MTs and activates mitochondria. In conclusion, we describe the existence of a specialized cellular network connecting the cellular energetic status and MT dynamics to coordinate the functioning of LDs and mitochondria during nutrient scarcity.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Animais , Células COS , Chlorocebus aethiops , Oxirredução , Tirosina/metabolismo , Células VeroRESUMO
Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of "emerging" LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage.
Assuntos
Coenzima A Ligases/fisiologia , Retículo Endoplasmático/metabolismo , Metabolismo dos Lipídeos , Animais , Células COS , Caveolina 1/química , Chlorocebus aethiops , Coenzima A Ligases/análise , Coenzima A Ligases/metabolismo , Retículo Endoplasmático/ultraestrutura , Proteínas de Fluorescência Verde/análise , Espaço Intracelular , Metiltransferases/química , Engenharia de Proteínas , Sinais Direcionadores de ProteínasRESUMO
Lipid droplets (LDs) are dynamic organelles that collect, store, and supply lipids [1]. LDs have a central role in the exchange of lipids occurring between the cell and the environment and provide cells with substrates for energy metabolism, membrane synthesis, and production of lipid-derived molecules such as lipoproteins or hormones. However, lipid-derived metabolites also cause progressive lipotoxicity [2], accumulation of reactive oxygen species (ROS), endoplasmic reticulum stress, mitochondrial malfunctioning, and cell death [2]. Intracellular accumulation of LDs is a hallmark of prevalent human diseases, including obesity, steatosis, diabetes, myopathies, and arteriosclerosis [3]. Indeed, nonalcoholic fatty liver disease is the most common cause of abnormal hepatic function among adults [4, 5]. Lipotoxicity gradually promotes cellular ballooning and disarray, megamitochondria, accumulation of Mallory's hyaline in hepatocytes, and inflammation, fibrosis, and cirrhosis in the liver. Here, using confocal microscopy, serial-block-face scanning electron microscopy, and flow cytometry, we show that LD accumulation is heterogeneous within a cell population and follows a positive skewed distribution. Lipid availability and fluctuations in biochemical networks controlling lipolysis, fatty acid oxidation, and protein synthesis contribute to cell-to-cell heterogeneity. Critically, this reversible variability generates a subpopulation of cells that effectively collect and store lipids. This high-lipid subpopulation accumulates more LDs and more ROS and reduces the risk of lipotoxicity to the population without impairing overall lipid homeostasis, since high-lipid cells can supply stored lipids to the other cells. In conclusion, we demonstrate fat storage compartmentalization within a cell population and propose that this is a protective social organization to reduce lipotoxicity.
Assuntos
Hepatócitos/citologia , Metabolismo dos Lipídeos , Lipídeos/química , Animais , Compostos de Boro/metabolismo , Ácidos Graxos/metabolismo , Citometria de Fluxo , Hepatócitos/metabolismo , Lipídeos/fisiologia , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismoRESUMO
Caveolins (CAVs) are essential components of caveolae, plasma membrane invaginations with reduced fluidity, reflecting cholesterol accumulation. CAV proteins bind cholesterol, and CAV's ability to move between cellular compartments helps control intracellular cholesterol fluxes. In humans, CAV1 mutations result in lipodystrophy, cell transformation, and cancer. CAV1 gene-disrupted mice exhibit cardiovascular diseases, diabetes, cancer, atherosclerosis, and pulmonary fibrosis. The mechanism or mechanisms underlying these disparate effects are unknown, but our past work suggested that CAV1 deficiency might alter metabolism: CAV1(-/-) mice exhibit impaired liver regeneration unless supplemented with glucose, suggesting systemic inefficiencies requiring additional metabolic intermediates. Establishing a functional link between CAV1 and metabolism would provide a unifying theme to explain these myriad pathologies. Here we demonstrate that impaired proliferation and low survival with glucose restriction is a shortcoming of CAV1-deficient cells caused by impaired mitochondrial function. Without CAV1, free cholesterol accumulates in mitochondrial membranes, increasing membrane condensation and reducing efficiency of the respiratory chain and intrinsic antioxidant defense. Upon activation of oxidative phosphorylation, this promotes accumulation of reactive oxygen species, resulting in cell death. We confirm that this mitochondrial dysfunction predisposes CAV1-deficient animals to mitochondrial-related diseases such as steatohepatitis and neurodegeneration.