RESUMEN
We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.
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Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Piperazinas , Tiazinas , Animales , Ratones , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Gránulos de Estrés , Mutaciones Letales SintéticasRESUMEN
IMPORTANCE: Staphylococcus aureus uses numerous strategies to survive and persist in the intracellular environment of professional phagocytes, including modulation of the SUMOylation process. This study aims to understand how S. aureus alters host SUMOylation to enhance its intracellular survival in professional phagocytes. Our results indicate that S. aureus strain Newman utilizes PtpA-driven phosphorylation to decrease the amount of SUMOylated proteins in murine macrophages to facilitate its survival in this immune cell type.
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Proteínas Tirosina Fosfatasas , Staphylococcus aureus , Sumoilación , Animales , Ratones , Macrófagos , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Staphylococcus aureus/metabolismo , Tirosina/metabolismo , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiologíaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been widely studied at multiomics level. However, little is known about its specific ubiquitination, a major post-translational modification (PTM). As PTMs regulate the final function of any gene, we decided to establish the ubiquitination profiles of 60 PDAC. METHODS: We used specific proteomic tools to establish the ubiquitin dependent proteome (ubiquitinome) of frozen PDXs (Patients' derived xenographs). Then, we performed bioinformatics analysis to identify the possible associations of these ubiquitination profiles with tumour phenotype, patient survival and resistance to chemotherapies. Finally, we used proximity ligation assays (PLA) to detect and quantify the ubiquitination level of one identified marker. FINDINGS: We identified 38 ubiquitination site profiles correlating with the transcriptomic phenotype of tumours and four had notable prognostic capabilities. Seventeen ubiquitination profiles displayed potential theranostic marker for gemcitabine, seven for 5-FU, six for oxaliplatin and thirteen for irinotecan. Using PLA, we confirmed the use of one ubiquitination profile as a drug-response marker, directly on paraffin embedded tissues, supporting the possible application of these biomarkers in the clinical setting. INTERPRETATION: These findings bring new and important insights on the relationship between ubiquitination levels of proteins and different molecular and clinical features of PDAC patients. Markers identified in this study could have a potential application in clinical settings to help to predict response to chemotherapies thereby allowing the personalization of treatments. FUNDING: Fondation ARC (PJA 20181208270 and PGA 12021010002840_3562); INCa; Canceropôle PACA; DGOS; Amidex Foundation; Fondation de France; and INSERM.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Pronóstico , Medicina de Precisión , Proteómica , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Ubiquitinación , Neoplasias PancreáticasRESUMEN
Proteomic, cellular and biochemical analysis of the stress protein NUPR1 reveals that it binds to PARP1 into the nucleus and inhibits PARP1 activity in vitro. Mutations on residues Ala33 or Thr68 of NUPR1 or treatment with its inhibitor ZZW-115 inhibits this effect. PARylation induced by 5-fluorouracil (5-FU) treatment is strongly enhanced by ZZW-115 and associated with a decrease of NAD+/NADH ratio and rescued by the PARP inhibitor olaparib. Cell death induced by ZZW-115 treatment of pancreas cancer-derived cells is rescued by olaparib and improved with PARG inhibitor PDD00017273. The mitochondrial catastrophe induced by ZZW-115 treatment or by genetic inactivation of NUPR1 is associated to a hyperPARylation of the mitochondria, disorganization of the mitochondrial network, mitochondrial membrane potential decrease, and with increase of superoxide production, intracellular level of reactive oxygen species (ROS) and cytosolic levels of Ca2+. These features are rescued by olaparib or NAD+ precursor nicotinamide mononucleotide in a dose-dependent manner and partially by antioxidants treatments. In conclusion, inactivation of NUPR1 induces a hyperPARylation, which in turn, induces a mitochondrial catastrophe and consequently a cell death through a non-canonical Parthanatos, since apoptosis inducing-factor (AIF) is not translocated out of the mitochondria.
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NAD , Tiazinas , Muerte Celular , NAD/metabolismo , Piperazinas , ProteómicaRESUMEN
Staphylococcus aureus is a commensal bacterium that causes severe infections in soft tissue and the bloodstream. During infection, S. aureus manipulates host cell response to facilitate its own replication and dissemination. Here, we show that S. aureus significantly decreases the level of SUMOylation, an essential post-translational modification, in infected macrophages 24 h post-phagocytosis. The reduced level of SUMOylation correlates with a decrease in the SUMO-conjugating enzyme Ubc9. The over-expression of SUMO proteins in macrophages impaired bacterial intracellular proliferation and the inhibition of SUMOylation with ML-792 increased it. Together, these findings demonstrated for the first time the role of host SUMOylation response toward S. aureus infection.
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Interacciones Microbiota-Huesped/inmunología , Macrófagos/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Animales , Macrófagos/citología , Ratones , Células RAW 264.7 , Sumoilación , Enzimas Ubiquitina-Conjugadoras/inmunologíaRESUMEN
Endothelial-mesenchymal transition (EndMT) is an important source of cancer-associated fibroblasts (CAFs), which facilitates tumour progression. PDAC is characterised by abundant CAFs and tumour necrosis factor-α (TNF-α). Here, we show that TNF-α strongly induces human endothelial cells to undergo EndMT. Interestingly, TNF-α strongly downregulates the expression of the endothelial receptor TIE1, and reciprocally TIE1 overexpression partially prevents TNF-α-induced EndMT, suggesting that TNF-α acts, at least partially, through TIE1 regulation in this process. We also show that TNF-α-induced EndMT is reversible. Furthermore, TNF-α treatment of orthotopic mice resulted in an important increase in the stroma, including CAFs. Finally, secretome analysis identified TNFSF12, as a regulator that is also present in PDAC patients. With the aim of restoring normal angiogenesis and better access to drugs, our results support the development of therapies targeting CAFs or inducing the EndMT reversion process in PDAC.
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Fibroblastos Asociados al Cáncer/efectos de los fármacos , Carcinoma Ductal Pancreático/patología , Células Endoteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Pancreáticas/patología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Células Cultivadas , Citocina TWEAK/genética , Citocina TWEAK/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Masculino , Ratones Transgénicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor TIE-1/genética , Receptor TIE-1/metabolismo , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismoRESUMEN
BACKGROUND: Although significant advances have been made recently to characterize the biology of pancreatic ductal adenocarcinoma (PDAC), more efforts are needed to improve our understanding and to face challenges related to the aggressiveness, high mortality rate and chemoresistance of this disease. METHODS: In this study, we perform the metabolomics profiling of 77 PDAC patient-derived tumor xenografts (PDTX) to investigate the relationship of metabolic profiles with overall survival (OS) in PDAC patients, tumor phenotypes and resistance to five anticancer drugs (gemcitabine, oxaliplatin, docetaxel, SN-38 and 5-Fluorouracil). FINDINGS: We identified a metabolic signature that was able to predict the clinical outcome of PDAC patients (p < 0.001, HR=2.68 [95% CI: 1.5-4.9]). The correlation analysis showed that this metabolomic signature was significantly correlated with the PDAC molecular gradient (PAMG) (R = 0.44 and p < 0.001) indicating significant association to the transcriptomic phenotypes of tumors. Resistance score established, based on growth rate inhibition metrics using 35 PDTX-derived primary cells, allowed to identify several metabolites related to drug resistance which was globally accompanied by accumulation of several diacy-phospholipids and decrease in lysophospholipids. Interestingly, targeting glycerophospholipid synthesis improved sensitivity to the three tested cytotoxic drugs indicating that interfering with metabolism could be a promising therapeutic strategy to overcome the challenging resistance of PDAC. INTERPRETATION: In conclusion, this study shows that the metabolomic profile of pancreatic PDTX models is strongly associated to clinical outcome, transcriptomic phenotypes and drug resistance. We also showed that targeting the lipidomic profile could be used in combinatory therapies against chemoresistance in PDAC.
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Adenocarcinoma/metabolismo , Resistencia a Antineoplásicos , Metaboloma , Metabolómica , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/etiología , Adenocarcinoma/mortalidad , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biología Computacional/métodos , Bases de Datos Genéticas , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Metabolómica/métodos , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/etiología , Neoplasias Pancreáticas/mortalidad , FenotipoRESUMEN
Nuclear protein 1 (NUPR1) is a stress response protein overexpressed upon cell injury in virtually all organs including the exocrine pancreas. Despite NUPR1's well-established role in the response to cell stress, the molecular and structural machineries triggered by NUPR1 activation remain largely debated. In this study, we uncover a new role for NUPR1, participating in the unfolded protein response (UPR) and the integrated stress response. Biochemical results and ultrastructural morphological observations revealed alterations in the UPR of acinar cells of germline-deleted NUPR1 murine models, consistent with the inability to restore general protein synthesis after stress induction. Bioinformatic analysis of NUPR1-interacting partners showed significant enrichment in translation initiation factors, including eukaryotic initiation factor (eIF) 2α. Co-immunoprecipitation and proximity ligation assays confirmed the interaction between NUPR1 and eIF2α and its phosphorylated form (p-eIF2α). Furthermore, our data suggest loss of NUPR1 in cells results in maintained eIF2α phosphorylation and evaluation of nascent proteins by click chemistry revealed that NUPR1-depleted PANC-1 cells displayed a slower poststress protein synthesis recovery when compared to wild-type. Combined, these data propose a novel role for NUPR1 in the integrated stress response pathway, at least partially through promoting efficient PERK branch activity and resolution through a unique interaction with eIF2α.
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Proteínas de Unión al ADN/genética , Estrés del Retículo Endoplásmico/genética , Factor 2 Eucariótico de Iniciación/genética , Regulación de la Expresión Génica , Proteínas de Neoplasias/genética , Páncreas/metabolismo , Respuesta de Proteína Desplegada/genética , Células Acinares/metabolismo , Células Acinares/ultraestructura , Animales , Western Blotting , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Ratones Noqueados , Microscopía Electrónica de Transmisión , Proteínas de Neoplasias/metabolismo , Páncreas/citología , Páncreas/ultraestructura , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Carcinoma Ductal Pancreático/tratamiento farmacológico , Núcleo Celular/metabolismo , Daño del ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Piperazinas/farmacología , Tiazinas/farmacología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Núcleo Celular/efectos de los fármacos , Proliferación Celular , Reparación del ADN , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transporte de Proteínas , Sumoilación , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Biomarkers and effective therapeutic agents to improve the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) are urgently required. We aimed to analyze the prognostic value and mechanistic action of miR-93 in PDAC. Correlation of miR-93 tumor levels from 83 PDAC patients and overall survival (OS) was analyzed by Kaplan-Meier. MiR-93 depletion in PANC-1 and MIA PaCa-2 cells was achieved by CRISPR/Cas9 and miR-93 overexpression in HPDE cells by retroviral transduction. Cell proliferation, migration and invasion, cell cycle analysis, and in vivo tumor xenografts in nude mice were assessed. Proteomic analysis by mass spectrometry and western-blot was also performed. Finally, miR-93 direct binding to candidate mRNA targets was evaluated by luciferase reporter assays. High miR-93 tumor levels are significantly correlated with a worst prognosis in PDAC patients. MiR-93 abolition altered pancreatic cancer cells phenotype inducing a significant increase in cell size and a significant decrease in cell invasion and proliferation accompanied by a G2/M arrest. In vivo, lack of miR-93 significantly impaired xenograft tumor growth. Conversely, miR-93 overexpression induced a pro-tumorigenic behavior by significantly increasing cell proliferation, migration, and invasion. Proteomic analysis unveiled a large group of deregulated proteins, mainly related to G2/M phase, microtubule dynamics, and cytoskeletal remodeling. CRMP2, MAPRE1, and YES1 were confirmed as direct targets of miR-93. MiR-93 exerts oncogenic functions by targeting multiple genes involved in microtubule dynamics at different levels, thus affecting the normal cell division rate. MiR-93 or its direct targets (CRMP2, MAPRE1, or YES1) are new potential therapeutic targets for PDAC.
RESUMEN
Cancer cells activate stress-response mechanisms to adapt themselves to a variety of stressful conditions. Among these protective mechanisms, those controlled by the stress-induced nuclear protein 1 (NUPR1 ) belong to the most conserved ones. NUPR1 is an 82-residue-long, monomeric, basic and intrinsically disordered protein (IDP), which was found to be invariably overexpressed in some, if not all, cancer tissues. Remarkably, we and others have previously showed that genetic inactivation of the Nupr1 gene antagonizes the growth of pancreatic cancer as well as several other tumors. With the use of a multidisciplinary strategy by combining biophysical, biochemical, bioinformatic, and biological approaches, a trifluoperazine-derived compound, named ZZW-115, has been identified as an inhibitor of the NUPR1 functions. The anticancer activity of the ZZW-115 was first validated on a large panel of cancer cells. Furthermore, ZZW-115 produced a dose-dependent tumor regression of the tumor size in xenografted mice. Mechanistically, we have demonstrated that NUPR1 binds to several importins. Because ZZW-115 binds NUPR1 through the region around the amino acid Thr68, which is located into the nuclear location signal (NLS) region of the protein, we demonstrated that treatment with ZZW-115 inhibits completely the translocation of NUPR1 from the cytoplasm to the nucleus by competing with importins.
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Adenocarcinoma/tratamiento farmacológico , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Fenotiazinas/uso terapéutico , Adenocarcinoma/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias Pancreáticas/metabolismo , Fenotiazinas/farmacología , Transporte de Proteínas/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.
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Procesamiento Proteico-Postraduccional , Ubiquitina/metabolismo , Línea Celular , Cromatografía Liquida , Humanos , Programas Informáticos , Espectrometría de Masas en TándemRESUMEN
The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is mainly due to its rapidly acquired resistance to all conventional treatments. Despite drug-specific mechanisms of resistance, none explains how these cells resist the stress induced by any kind of anticancer treatment. Activation of stress-response pathways relies on the post-translational modifications (PTMs) of involved proteins. Among all PTMs, those mediated by the ubiquitin family of proteins play a central role. Our aim was to identify alterations of ubiquitination, neddylation, and sumoylation associated with the multiresistant phenotype and demonstrate their implications in the survival of PDAC cells undergoing treatment. This approach pointed at an alteration of promyelocytic leukemia (PML) protein sumoylation associated with both gemcitabine and oxaliplatin resistance. We could show that this alteration of PML sumoylation is part of a general mechanism of drug resistance, which in addition involves the abnormal activation of NF-κB and cAMP response element binding pathways. Importantly, using patient-derived tumors and cell lines, we identified a correlation between the levels of PML expression and sumoylation and the sensitivity of tumors to anticancer treatments.-Swayden, M., Alzeeb, G., Masoud, R., Berthois, Y., Audebert, S., Camoin, L., Hannouche, L., Vachon, H., Gayet, O., Bigonnet, M., Roques, J., Silvy, F., Carrier, A., Dusetti, N., Iovanna, J. L., Soubeyran, P. PML hyposumoylation is responsible for the resistance of pancreatic cancer.
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Resistencia a Antineoplásicos , Proteína de la Leucemia Promielocítica/metabolismo , Sistemas de Mensajero Secundario , Antineoplásicos/farmacología , Línea Celular Tumoral , AMP Cíclico/genética , AMP Cíclico/metabolismo , Células HEK293 , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteína de la Leucemia Promielocítica/genética , SumoilaciónRESUMEN
Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because they have dynamical structure features and conventional drug design is not applicable. NUPR1 is an IDP playing an important role in pancreatic cancer. We previously reported that Trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumors growth. Unfortunately, TFP showed strong central nervous system side-effects. In this work, we undertook a multidisciplinary approach to optimize TFP, based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical and biological evaluations. A family of TFP-derived compounds was produced and the most active one, named ZZW-115, showed a dose-dependent tumor regression with no neurological effects and induced cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets.
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Antineoplásicos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Necroptosis/efectos de los fármacos , Proteínas de Neoplasias , Neoplasias/tratamiento farmacológico , Trifluoperazina , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Hep G2 , Humanos , Células Jurkat , Ratones , Ratones Desnudos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Células PC-3 , Trifluoperazina/análogos & derivados , Trifluoperazina/síntesis química , Trifluoperazina/química , Trifluoperazina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To date, PDAC remains the cancer having the worst prognosis with mortality rates constantly on the rise. Efficient cures are still absent, despite all attempts to understand the aggressive physiopathology underlying this disease. A major stumbling block is the outdated preclinical modeling strategies applied in assessing effectiveness of novel anticancer therapeutics. Current in vitro preclinical models have a low fidelity to mimic the exact architectural and functional complexity of PDAC tumor found in human set, due to the lack of major components such as immune system and tumor microenvironment with its associated chemical and mechanical signals. The existing PDAC preclinical platforms are still far from being reliable and trustworthy to guarantee the success of a drug in clinical trials. Therefore, there is an urgent demand to innovate novel in vitro preclinical models that mirrors with precision tumor-microenvironment interface, pressure of immune system, and molecular and morphological aspects of the PDAC normally experienced within the living organ. This review outlines the traditional preclinical models of PDAC namely 2D cell lines, genetically engineered mice, and xenografts, and describing the present famous approach of 3D organoids. We offer a detailed narration of the pros and cons of each model system. Finally, we suggest the incorporation of two off-center newly born techniques named 3D bio-printing and organs-on-chip and discuss the potentials of swine models and in silico tools, as powerful new tools able to transform PDAC preclinical modeling to a whole new level and open new gates in personalized medicine.
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Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest forms of cancer. A major reason for this situation is the fact that these tumors are already resistant or become rapidly resistant to all conventional therapies. Like any transformation process, initiation and development of PDCA are driven by a well known panel of genetic alterations, few of them are shared with most cancers, but many mutations are specific to PDAC and are partially responsible for the great inter-tumor heterogeneity. Importantly, this knowledge has been inefficient in predicting response to anticancer therapy, or in establishing diagnosis and prognosis. Hence, the pre-existing or rapidly acquired resistance of pancreatic cancer cells to therapeutic drugs rely on other parameters and features developed by the cells and/or the micro-environment, that are independent of their genetic profiles. This review sheds light on all major phenotypic, non genetic, alterations known to play important roles in PDAC cells resistance to treatments and therapeutic escape.
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The microRNA-200 (miR-200) family is frequently down-regulated in tumors, including pancreatic adenocarcinomas (PDACs). In this study we have examined the mechanisms involved in the loss of miR-200s in tumoral pancreatic cells. Whereas miR-200 gene promoters appear methylated in mature miR-200 deficient cell lines, miR-200 precursors are detected in nuclear but not cytoplasmic compartment of these cells, indicating that promoter hypermethylation is not sufficient to explain the deficit of mature miR-200s. The ribonuclease Monocyte Chemotactic Protein-induced Protein-1 (MCPiP1) may counteract Dicer1 in miRNA maturation process. MCPiP1/Dicer1 mRNA and protein ratios appear higher in miR-200 deficient compared to miR-200 proficient cells, suggesting that MCPiP1 may compete with Dicer1 in mature miR-200 deficient cells. Inhibition of MCPiP1 allows the detection of miR-200 precursors in cytoplasm of miR-200 deficient cells, confirming its involvement in the loss of miR-200s. Also, reversion of MCPiP1/Dicer1 ratio by over-expression of Dicer1 in miR-200 deficient cells leads to the recovery of mature miR-200s. Finally, whereas human malignant pancreatic tissues (PDACs) express lower miR-200 levels than non malignant tissues (non-MPDs), MCPiP1/Dicer1 ratio appears higher in PDACs, when compared to non-MPDs, supporting the hypothesis that MCPiP1/Dicer1 ratio is determinant in regulating miR-200 maturation process in a subset of tumoral pancreatic cells.
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It was already described that genetic inhibition of NUPR1 induces tumor growth arrest. In this paper we studied the metabolism changes after NUPR1 downregulation in pancreatic cancer cells, which results in a significant decrease of OXPHOS activity with a concomitant lower ATP production which precedes the necrotic cell death. We demonstrated that NUPR1 downregulation induces a mitochondrial failure with a loss of the mitochondrial membrane potential, a strong increase in ROS production and a concomitant relocalization of mitochondria to the vicinity of the endoplasmic reticulum (ER). In addition, the transcriptomic analysis of NUPR1-deficient cells shows a decrease in the expression of some ER stress response-associated genes. Indeed, in ER stressors-treated cells with thapsigargin, brefeldin A or tunicamycin, a greater increase in necrosis and decrease of ATP content was observed in NUPR1-defficent cells. Finally, in vivo experiments, using acute pancreatitis which induces ER stress as well as NUPR1 activation, we observed that NUPR1 expression protects acinar cells from necrosis in mice. Importantly, we also report that the cell death observed after knocking-down NUPR1 expression is completely reversed by incubation with Necrostatin-1, but not by inhibiting caspase activity with Z-VAD-FMK. Altogether, these data enable us to describe a model in which inactivation of NUPR1 in pancreatic cancer cells results in an ER stress that induces a mitochondrial malfunction, a deficient ATP production and, as consequence, the cell death mediated by a programmed necrosis.
Asunto(s)
Muerte Celular , Proteínas de Unión al ADN/fisiología , Estrés del Retículo Endoplásmico , Mitocondrias/patología , Necrosis , Proteínas de Neoplasias/fisiología , Neoplasias Pancreáticas/patología , Pancreatitis/patología , Células Acinares , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Mitofagia , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilación Oxidativa , Páncreas/metabolismo , Páncreas/patología , Neoplasias Pancreáticas/metabolismo , Pancreatitis/metabolismoRESUMEN
In this study, our aim is to assess the role played by autophagy and its inhibition in the different PDAC cellular compartments, and its involvement in chemo-resistance using primary human pancreatic cancer-derived cells (PCC) and Cancer Associated Fibroblasts (CAF). Autophagy flux, as measured by LC3-I and -II in the presence of Chloroquine, showed a variable level in PCC and CAFs. We found no correlation between autophagy level and degree of tumor differentiation. Association of Chloroquine with gemcitabine, 5FU, oxaliplatin, irinotecan and docetaxel revealed that its effect on survival is cell- and drug-dependent in vitro and in vivo. In addition, we demonstrated that autophagy in CAFs can play an important role in sensitizing PDAC to anticancer treatments since its inhibition increased the resistance of PCCs to gemcitabine. In conclusion, this work clearly shows a heterogeneity in the effect of Chloroquine and highlights a role of CAFs autophagy in sensitizing tumors to treatments. It also reveals that the role of autophagy is more complex than expected in PDAC as well as its sensitivity to treatments.
RESUMEN
Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (â¼10 µM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins.