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1.
Cell Rep ; 43(6): 114281, 2024 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-38805395

RESUMEN

Survival from UV-induced DNA lesions relies on nucleotide excision repair (NER) and the Mec1ATR DNA damage response (DDR). We study DDR and NER in aging cells and find that old cells struggle to repair DNA and activate Mec1ATR. We employ pharmacological and genetic approaches to rescue DDR and NER during aging. Conditions activating Snf1AMPK rescue DDR functionality, but not NER, while inhibition of the TORC1-Sch9S6K axis restores NER and enhances DDR by tuning PP2A activity, specifically in aging cells. Age-related repair deficiency depends on Snf1AMPK-mediated phosphorylation of Sch9S6K on Ser160 and Ser163. PP2A activity in old cells is detrimental for DDR and influences NER by modulating Snf1AMPK and Sch9S6K. Hence, the DDR and repair pathways in aging cells are influenced by the metabolic tuning of opposing AMPK and TORC1 networks and by PP2A activity. Specific Sch9S6K phospho-isoforms control DDR and NER efficiency, specifically during aging.

2.
Sci Transl Med ; 16(736): eadf9874, 2024 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-38416843

RESUMEN

Targeting aromatase deprives ER+ breast cancers of estrogens and is an effective therapeutic approach for these tumors. However, drug resistance is an unmet clinical need. Lipidomic analysis of long-term estrogen-deprived (LTED) ER+ breast cancer cells, a model of aromatase inhibitor resistance, revealed enhanced intracellular lipid storage. Functional metabolic analysis showed that lipid droplets together with peroxisomes, which we showed to be enriched and active in the LTED cells, controlled redox homeostasis and conferred metabolic adaptability to the resistant tumors. This reprogramming was controlled by acetyl-CoA-carboxylase-1 (ACC1), whose targeting selectively impaired LTED survival. However, the addition of branched- and very long-chain fatty acids reverted ACC1 inhibition, a process that was mediated by peroxisome function and redox homeostasis. The therapeutic relevance of these findings was validated in aromatase inhibitor-treated patient-derived samples. Last, targeting ACC1 reduced tumor growth of resistant patient-derived xenografts, thus identifying a targetable hub to combat the acquisition of estrogen independence in ER+ breast cancers.


Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Inhibidores de la Aromatasa/farmacología , Inhibidores de la Aromatasa/uso terapéutico , Peroxisomas/metabolismo , Peroxisomas/patología , Acetil-CoA Carboxilasa , Gotas Lipídicas/metabolismo , Gotas Lipídicas/patología , Línea Celular Tumoral , Estrógenos/metabolismo , Resistencia a Antineoplásicos
3.
Genome Med ; 16(1): 15, 2024 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-38243308

RESUMEN

BACKGROUND: Immunotherapy based on checkpoint inhibitors is highly effective in mismatch repair deficient (MMRd) colorectal cancer (CRC). These tumors carry a high number of mutations, which are predicted to translate into a wide array of neoepitopes; however, a systematic classification of the neoantigen repertoire in MMRd CRC is lacking. Mass spectrometry peptidomics has demonstrated the existence of MHC class I associated peptides (MAPs) originating from non-coding DNA regions. Based on these premises we investigated DNA genomic regions responsible for generating MMRd-induced peptides. METHODS: We exploited mouse CRC models in which the MMR gene Mlh1 was genetically inactivated. Isogenic cell lines CT26 Mlh1+/+ and Mlh1-/- were inoculated in immunocompromised and immunocompetent mice. Whole genome and RNA sequencing data were generated from samples obtained before and after injection in murine hosts. First, peptide databases were built from transcriptomes of isogenic cell lines. We then compiled a database of peptides lost after tumor cells injection in immunocompetent mice, likely due to immune editing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matched next-generation sequencing databases were employed to identify the DNA regions from which the immune-targeted MAPs originated. Finally, we adopted in vitro T cell assays to verify whether MAP-specific T cells were part of the in vivo immune response against Mlh1-/- cells. RESULTS: Whole genome sequencing analyses revealed an unbalanced distribution of immune edited alterations across the genome in Mlh1-/- cells grown in immunocompetent mice. Specifically, untranslated (UTR) and coding regions exhibited the largest fraction of mutations leading to highly immunogenic peptides. Moreover, the integrated computational and LC-MS/MS analyses revealed that MAPs originate mainly from atypical translational events in both Mlh1+/+ and Mlh1-/- tumor cells. In addition, mutated MAPs-derived from UTRs and out-of-frame translation of coding regions-were highly enriched in Mlh1-/- cells. The MAPs trigger T-cell activation in mice primed with Mlh1-/- cells. CONCLUSIONS: Our results suggest that-in comparison to MMR proficient CRC-MMRd tumors generate a significantly higher number of non-canonical mutated peptides able to elicit T cell responses. These results reveal the importance of evaluating the diversity of neoepitope repertoire in MMRd tumors.


Asunto(s)
Neoplasias Encefálicas , Neoplasias del Colon , Neoplasias Colorrectales , Síndromes Neoplásicos Hereditarios , Animales , Ratones , Reparación de la Incompatibilidad de ADN/genética , Cromatografía Liquida , Espectrometría de Masas en Tándem , Neoplasias Colorrectales/patología , Péptidos , Antígenos de Histocompatibilidad Clase I/genética , ADN
4.
Cell Death Dis ; 15(1): 28, 2024 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-38199984

RESUMEN

The tumor microenvironment is a complex ecosystem that plays a critical role in cancer progression and treatment response. Recently, extracellular amyloid fibrils have emerged as novel components of the tumor microenvironment; however, their function remains elusive. In this study, we establish a direct connection between the presence of amyloid fibrils in the secretome and the activation of YAP, a transcriptional co-activator involved in cancer proliferation and drug resistance. Furthermore, we uncover a shared mechano-signaling mechanism triggered by amyloid fibrils in both melanoma and pancreatic ductal adenocarcinoma cells. Our findings highlight the crucial role of the glycocalyx protein Agrin which binds to extracellular amyloid fibrils and acts as a necessary factor in driving amyloid-dependent YAP activation. Additionally, we reveal the involvement of the HIPPO pathway core kinase LATS1 in this signaling cascade. Finally, we demonstrate that extracellular amyloid fibrils enhance cancer cell migration and invasion. In conclusion, our research expands our knowledge of the tumor microenvironment by uncovering the role of extracellular amyloid fibrils in driving mechano-signaling and YAP activation. This knowledge opens up new avenues for developing innovative strategies to modulate YAP activation and mitigate its detrimental effects during cancer progression.


Asunto(s)
Melanoma , Neoplasias Pancreáticas , Humanos , Amiloide , Ecosistema , Transducción de Señal , Neoplasias Pancreáticas/genética , Microambiente Tumoral
5.
Cell Rep ; 42(12): 113555, 2023 12 26.
Artículo en Inglés | MEDLINE | ID: mdl-38088930

RESUMEN

Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.


Asunto(s)
Ataxia Telangiectasia , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Cromatina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteómica , Proteínas de Unión al ADN/metabolismo , Fosforilación , Daño del ADN , Citoesqueleto/metabolismo
6.
Sci Adv ; 9(37): eadh4184, 2023 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-37713487

RESUMEN

Cancers feature substantial intratumoral heterogeneity of genetic and phenotypically distinct lineages. Although interactions between coexisting lineages are emerging as a potential contributor to tumor evolution, the extent and nature of these interactions remain largely unknown. We postulated that tumors develop ecological interactions that sustain diversity and facilitate metastasis. Using a combination of fluorescent barcoding, mathematical modeling, metabolic analysis, and in vivo models, we show that the Allee effect, i.e., growth dependency on population size, is a feature of tumor lineages and that cooperative ecological interactions between lineages alleviate the Allee barriers to growth in a model of triple-negative breast cancer. Soluble metabolite exchange formed the basis for these cooperative interactions and catalyzed the establishment of a polyclonal community that displayed enhanced metastatic dissemination and outgrowth in xenograft models. Our results highlight interclonal metabolite exchange as a key modulator of tumor ecology and a contributing factor to overcoming Allee effect-associated growth barriers to metastasis.


Asunto(s)
Colorantes , Neoplasias de la Mama Triple Negativas , Humanos , Animales , Modelos Animales de Enfermedad , Densidad de Población
7.
J Neurochem ; 166(2): 346-366, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37303123

RESUMEN

Astrocytes associate with amyloid plaques in Alzheimer's disease (AD). Astrocytes react to changes in the brain environment, including increasing concentrations of amyloid-ß (Aß). However, the precise response of astrocytes to soluble small Aß oligomers at concentrations similar to those present in the human brain has not been addressed. In this study, we exposed astrocytes to media from neurons that express the human amyloid precursor protein (APP) transgene with the double Swedish mutation (APPSwe), and which contains APP-derived fragments, including soluble human Aß oligomers. We then used proteomics to investigate changes in the astrocyte secretome. Our data show dysregulated secretion of astrocytic proteins involved in the extracellular matrix and cytoskeletal organization and increase secretion of proteins involved in oxidative stress responses and those with chaperone activity. Several of these proteins have been identified in previous transcriptomic and proteomic studies using brain tissue from human AD and cerebrospinal fluid (CSF). Our work highlights the relevance of studying astrocyte secretion to understand the brain response to AD pathology and the potential use of these proteins as biomarkers for the disease.


Asunto(s)
Enfermedad de Alzheimer , Astrocitos , Humanos , Astrocitos/metabolismo , Proteómica , Secretoma , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo
8.
EMBO J ; 42(10): e112234, 2023 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-36970857

RESUMEN

The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.


Asunto(s)
Antivirales , COVID-19 , Humanos , Interferones/metabolismo , Fosfolípidos , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Viral , Proteínas de Unión al ARN/metabolismo , SARS-CoV-2/metabolismo , Internalización del Virus , Proteínas de la Membrana/metabolismo
9.
Cell Rep ; 42(3): 112215, 2023 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-36917609

RESUMEN

Drugs targeting microtubules rely on the mitotic checkpoint to arrest cell proliferation. The prolonged mitotic arrest induced by such drugs is followed by a G1 arrest. Here, we follow for several weeks the fate of G1-arrested human cells after treatment with nocodazole. We find that a small fraction of cells escapes from the arrest and resumes proliferation. These escaping cells experience reduced DNA damage and p21 activation. Cells surviving treatment are enriched for anti-apoptotic proteins, including Triap1. Increasing Triap1 levels allows cells to survive the first treatment with reduced DNA damage and lower levels of p21; accordingly, decreasing Triap1 re-sensitizes cells to nocodazole. We show that Triap1 upregulation leads to the retention of cytochrome c in the mitochondria, opposing the partial activation of caspases caused by nocodazole. In summary, our results point to a potential role of Triap1 upregulation in the emergence of resistance to drugs that induce prolonged mitotic arrest.


Asunto(s)
Apoptosis , Mitosis , Humanos , Nocodazol/farmacología , Regulación hacia Arriba , Proliferación Celular , Fase G1 , Péptidos y Proteínas de Señalización Intracelular/genética
10.
J Exp Clin Cancer Res ; 41(1): 34, 2022 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-35073946

RESUMEN

BACKGROUND: Acute myeloid leukemia (AML) is characterized by accumulation of aberrantly differentiated hematopoietic myeloid progenitor cells. The karyotyping-silent NUP98-NSD1 fusion is a molecular hallmark of pediatric AML and is associated with the activating FLT3-ITD mutation in > 70% of the cases. NUP98-NSD1 fusion protein promotes myeloid progenitor self-renewal in mice via unknown molecular mechanism requiring both the NUP98 and the NSD1 moieties. METHODS: We used affinity purification coupled to label-free mass spectrometry (AP-MS) to examine the effect of NUP98-NSD1 structural domain deletions on nuclear interactome binding. We determined their functional relevance in NUP98-NSD1 immortalized primary murine hematopoietic stem and progenitor cells (HSPC) by inducible knockdown, pharmacological targeting, methylcellulose assay, RT-qPCR analysis and/or proximity ligation assays (PLA). Fluorescence recovery after photobleaching and b-isoxazole assay were performed to examine the phase transition capacity of NUP98-NSD1 in vitro and in vivo. RESULTS: We show that NUP98-NSD1 core interactome binding is largely dependent on the NUP98 phenylalanine-glycine (FG) repeat domains which mediate formation of liquid-like phase-separated NUP98-NSD1 nuclear condensates. We identified condensate constituents including imitation switch (ISWI) family member SMARCA5 and BPTF (bromodomain PHD finger transcription factor), both members of the nucleosome remodeling factor complex (NURF). We validated the interaction with SMARCA5 in NUP98-NSD1+ patient cells and demonstrated its functional role in NUP98-NSD1/FLT3-ITD immortalized primary murine hematopoietic cells by genetic and pharmacological targeting. Notably, SMARCA5 inhibition did not affect NUP98-NSD1 condensates suggesting that functional activity rather than condensate formation per se is crucial to maintain the transformed phenotype. CONCLUSIONS: NUP98-NSD1 interacts and colocalizes on the genome with SMARCA5 which is an essential mediator of the NUP98-NSD1 transformation in hematopoietic cells. Formation of NUP98-NSD1 phase-separated nuclear condensates is not sufficient for the maintenance of transformed phenotype, which suggests that selective targeting of condensate constituents might represent a new therapeutic strategy for NUP98-NSD1 driven AML.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Hematopoyesis/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteómica/métodos , Animales , Humanos , Ratones
11.
Metabolites ; 11(11)2021 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-34822378

RESUMEN

Lipidomics is the comprehensive analysis of lipids in a given biological system. This investigation is often limited by the low amount and high complexity of biological samples, therefore highly sensitive lipidomics methods are required. Nanoflow-LC/MS offers extremely high sensitivity; however, it is challenging as a more demanding maintenance is often needed compared to conventional microflow-LC approaches. Here, we developed a sensitive and reproducible lipidomics LC method, termed Opti-nQL, which can be applied to any biological system. Opti-nQL has been validated with cellular lipid extracts of human and mouse origin and with different lipid extraction methods. Among the resulting 4000 detected features, 700 and even more unique lipid molecular species have been identified covering 16 lipid sub-classes, while 400 lipids were uniquely structure defined by MS/MS. These results were obtained by analyzing an amount of lipids extract equivalent to 40 ng of proteins, being highly suitable for low abundant samples. MS analysis showed that theOpti-nQL method increases the number of identified lipids, which is evidenced by injecting 20 times less material than in microflow based chromatography, being more reproducible and accurate thus enhancing robustness of lipidomics analysis.

12.
Methods Mol Biol ; 2228: 401-408, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33950505

RESUMEN

Many classes of key functional proteins such as transcription factors or cell cycle proteins are present in the proteome at a very low concentration. These low-abundance proteins are almost entirely invisible to systematic quantitative analysis by classical data dependent proteomics methods (DDA). Moreover, DDA runs in shotgun proteomics experiments are plenty of missing values among the replicates due to the stochastic nature of the acquisition method, thus hampering the robustness of the quantitative analysis. Here, we have overcome these obstacles designing a robust workflow named missing value monitoring (MvM) in order to follow low abundance proteins dynamics.


Asunto(s)
Proteínas/análisis , Proteoma , Proteómica , Espectrometría de Masas en Tándem , Animales , Cromatografía de Fase Inversa , Humanos , Proyectos de Investigación , Flujo de Trabajo
13.
Stem Cell Reports ; 16(6): 1478-1495, 2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-33989519

RESUMEN

Globoid cell leukodystrophy (GLD) is a rare neurodegenerative lysosomal storage disease caused by an inherited deficiency of ß-galactocerebrosidase (GALC). GLD pathogenesis and therapeutic correction have been poorly studied in patient neural cells. Here, we investigated the impact of GALC deficiency and lentiviral vector-mediated GALC rescue/overexpression in induced pluripotent stem cell (iPSC)-derived neural progenitors and neuronal/glial progeny obtained from two GLD patients. GLD neural progeny displayed progressive psychosine storage, oligodendroglial and neuronal defects, unbalanced lipid composition, and early activation of cellular senescence, depending on the disease-causing mutation. The partial rescue of the neural differentiation program upon GALC reconstitution and psychosine clearance suggests multiple mechanisms contributing to neural pathology in GLD. Also, the pathological phenotype associated to supraphysiological GALC levels highlights the need of regulated GALC expression for proper human neural commitment/differentiation. These data have important implications for establishing safe therapeutic strategies to enhance disease correction of GLD.


Asunto(s)
Galactosilceramidasa/genética , Galactosilceramidasa/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Oligodendroglía/metabolismo , Diferenciación Celular , Células Cultivadas , Predisposición Genética a la Enfermedad , Terapia Genética/métodos , Humanos , Fenotipo , Psicosina/metabolismo , Células Madre/metabolismo
14.
J Exp Clin Cancer Res ; 40(1): 147, 2021 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-33926496

RESUMEN

BACE1 and BACE2 belong to a class of proteases called ß-secretases involved in ectodomain shedding of different transmembrane substrates. These enzymes have been extensively studied in Alzheimer's disease as they are responsible for the processing of APP in neurotoxic Aß peptides. These proteases, especially BACE2, are overexpressed in tumors and correlate with poor prognosis. Recently, different research groups tried to address the role of BACE1 and 2 in cancer development and progression. In this review, we summarize the latest findings on ß-secretases in cancer, highlighting the mechanisms that build the rationale to propose inhibitors of these proteins as a new line of treatment for different tumor types.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Neoplasias/genética , Humanos , Neoplasias/patología , Transducción de Señal , Microambiente Tumoral
15.
J Nephrol ; 34(3): 739-751, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33398797

RESUMEN

BACKGROUND: Hypertension is a complex disease and is the major cause of cardiovascular complications. In the vast majority of individuals, the aetiology of elevated blood pressure (BP) cannot be determined, thus impairing optimized therapies and prognosis for individual patients. A more precise understanding of the molecular pathogenesis of hypertension remains a pressing priority for both basic and translational research. Here we investigated the effect of salt on naive hypertensive patients in order to better understand the salt intake-blood pressure relationship. METHODS: Patients underwent an acute saline infusion and were defined as salt-sensitive or salt-resistant according to mean blood pressure changes. Urinary proteome changes during the salt load test were analysed by a label-free quantitative proteomics approach. RESULTS: Our data show that salt-sensitive patients display equal sodium reabsorption as salt-resistant patients, as major sodium transporters show the same behaviour during the salt load. However, salt-sensitive patients regulate the renin angiotensin system (RAS) differently from salt-resistant patients, and upregulate proteins, as epidermal growth factor (EGF) and plasminogen activator, urokinase (PLAU), involved in the regulation of epithelial sodium channel ENaC activity. CONCLUSIONS: Salt-sensitive and salt-resistant subjects have similar response to a saline/volume infusion as detected by urinary proteome. However, we identified glutamyl aminopeptidase (ENPEP), PLAU, EGF and Xaa-Pro aminopeptidase 2 precursor XPNPEP2 as key molecules of salt-sensitivity, through modulation of ENaC-dependent sodium reabsorption along the distal tubule.


Asunto(s)
Hipertensión , Cloruro de Sodio Dietético , Presión Sanguínea , Humanos , Hipertensión/diagnóstico , Proteómica , Sodio , Cloruro de Sodio Dietético/efectos adversos
16.
STAR Protoc ; 1(3): 100162, 2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33377056

RESUMEN

Secretome analysis is crucial to unravel extracellular processes. However, secreted proteins are difficult to detect in mass-spectrometry-based analysis due to contamination of serum proteins deriving from cell culture media and to high glycosylation, which hampers tryptic digestion. Secret3D workflow is an optimized protocol for the global analysis of secretome from in vitro cultured cells. It allows efficient and robust protein identification and quantitation and provides information on putative N-glycosylation sites of the secreted proteins. For complete details on the use and execution of this protocol, please refer to Matafora et al. (2020).


Asunto(s)
Algoritmos , Proteómica/métodos , Secretoma/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Células Cultivadas , Glicosilación , Humanos , Péptidos/química , Péptidos/aislamiento & purificación , Proteolisis , Proteoma/metabolismo , Fracciones Subcelulares/metabolismo , Flujo de Trabajo
17.
Nat Commun ; 11(1): 4828, 2020 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-32973141

RESUMEN

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Núcleo Celular/metabolismo , Estrés Mecánico , Citoesqueleto de Actina , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Encéfalo , Cromatina , Citoplasma , Citoesqueleto/metabolismo , Daño del ADN , Ratones Noqueados , Metástasis de la Neoplasia , Neurogénesis , Membrana Nuclear/metabolismo
18.
EMBO Rep ; 21(9): e50446, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32749065

RESUMEN

Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes-associated protein (YAP). Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance. Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood. Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils. We also confirm the accumulation of amyloid-like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta-secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta-secretase inhibitors as potential therapeutic approach for metastatic melanoma.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Melanoma , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Amiloidogénicas , Humanos , Mecanotransducción Celular , Melanoma/tratamiento farmacológico , Melanoma/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
19.
Anal Chem ; 92(13): 8874-8882, 2020 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-32501676

RESUMEN

Metabolomics and lipidomics studies are becoming increasingly popular but available tools for automated data analysis are still limited. The major issue in untargeted metabolomics is linked to the lack of efficient ranking methods allowing accurate identification of metabolites. Herein, we provide a user-friendly open-source software, named SMfinder, for the robust identification and quantification of small molecules. The software introduces an MS2 false discovery rate approach, which is based on single spectral permutation and increases identification accuracy. SMfinder can be efficiently applied to shotgun and targeted analysis in metabolomics and lipidomics without requiring extensive in-house acquisition of standards as it provides accurate identification by using available MS2 libraries in instrument independent manner. The software, downloadable at www.ifom.eu/SMfinder, is suitable for untargeted, targeted, and flux analysis.


Asunto(s)
Lipidómica/métodos , Metabolómica/métodos , Interfaz Usuario-Computador , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Línea Celular Tumoral , Humanos , Lípidos/análisis , Metaboloma
20.
Cancers (Basel) ; 12(3)2020 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-32183295

RESUMEN

The long-term efficacy of the Epidermal Growth Factor Receptor (EGFR)-targeted antibody cetuximab in advanced colorectal cancer (CRC) patients is limited by the emergence of drug-resistant (persister) cells. Recent studies in other cancer types have shown that cells surviving initial treatment with targeted agents are often vulnerable to alterations in cell metabolism including oxidative stress. Vitamin C (VitC) is an antioxidant agent which can paradoxically trigger oxidative stress at pharmacological dose. Here we tested the hypothesis that VitC in combination with cetuximab could restrain the emergence of secondary resistance to EGFR blockade in CRC RAS/BRAF wild-type models. We found that addition of VitC to cetuximab impairs the emergence of drug persisters, limits the growth of CRC organoids, and significantly delays acquired resistance in CRC patient-derived xenografts. Mechanistically, proteomic and metabolic flux analysis shows that cetuximab blunts carbohydrate metabolism by blocking glucose uptake and glycolysis, beyond promoting slow but progressive ROS production. In parallel, VitC disrupts iron homeostasis and further increases ROS levels ultimately leading to ferroptosis. Combination of VitC and cetuximab orchestrates a synthetic lethal metabolic cell death program triggered by ATP depletion and oxidative stress, which effectively limits the emergence of acquired resistance to anti-EGFR antibodies. Considering that high-dose VitC is known to be safe in cancer patients, our findings might have clinical impact on CRC patients treated with anti-EGFR therapies.

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