RESUMO
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.
Assuntos
Interações entre Hospedeiro e Microrganismos/imunologia , Macrófagos/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Animais , Macrófagos/citologia , Camundongos , Células RAW 264.7 , Sumoilação , Enzimas de Conjugação de Ubiquitina/imunologiaRESUMO
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.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteína da Leucemia Promielocítica/metabolismo , Sistemas do Segundo Mensageiro , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , AMP Cíclico/genética , AMP Cíclico/metabolismo , Células HEK293 , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína da Leucemia Promielocítica/genética , SumoilaçãoRESUMO
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.
RESUMO
To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.
Assuntos
Endotélio Vascular/fisiologia , Regulação da Expressão Gênica/fisiologia , Histona Desacetilases/metabolismo , Neovascularização Fisiológica/fisiologia , Proteína Fosfatase 2/metabolismo , Grau de Desobstrução Vascular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Adesão Celular/fisiologia , Colágeno , Combinação de Medicamentos , Imunofluorescência , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Processamento de Imagem Assistida por Computador , Laminina , Microscopia Confocal , Proteoglicanas , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Grau de Desobstrução Vascular/genética , Peixe-ZebraRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive malignancy characterized by an excessive resistance to all known anticancer therapies, a still largely elusive phenomenon. To identify original mechanisms, we have explored the role of post-translational modifications (PTMs) mediated by members of the ubiquitin family. Although alterations of these pathways have been reported in different cancers, no methodical search for these kinds of anomalies has been performed so far. Therefore, we studied the ubiquitin-, Nedd8-, and SUMO1-specific proteomes of a pancreatic cancer cell line (MiaPaCa-2) and identified changes induced by gemcitabine, the standard PDAC's chemotherapeutic drug. These PTMs profiles contained both known major substrates of all three modifiers as well as original ones. Gemcitabine treatment altered the PTM profile of proteins involved in various biological functions, some known cancer associated genes, many potentially cancer-associated genes, and several cancer-signaling networks, including canonical and noncanonical WNT and PI3K/Akt/MTOR pathways. Some of these altered PTMs formed groups of functionally and physically associated proteins. Importantly, we could validate the gemcitabine-induced PTMs variations of relevant candidates and we could demonstrate the biological significance of such altered PTMs by studying in detail the sumoylation of SNIP1, one of these new targets.
Assuntos
Antineoplásicos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microscopia de Fluorescência , Proteína NEDD8 , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Espectrometria de Massas em Tandem , Ubiquitina/genética , Ubiquitinas/genética , Ubiquitinas/metabolismo , Proteína Wnt1/metabolismo , GencitabinaRESUMO
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.
Assuntos
Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Piperazinas , Tiazinas , Animais , Camundongos , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/genética , Caspase 3/genética , Caspase 3/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Grânulos de Estresse , Mutações Sintéticas LetaisRESUMO
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.
Assuntos
Proteínas Tirosina Fosfatases , Staphylococcus aureus , Sumoilação , Animais , Camundongos , Macrófagos , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Staphylococcus aureus/metabolismo , Tirosina/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologiaRESUMO
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.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Prognóstico , Medicina de Precisão , Proteômica , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Ubiquitinação , Neoplasias PancreáticasRESUMO
ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin.
Assuntos
Células Epiteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Isoformas de Proteínas/metabolismo , Junções Íntimas/metabolismo , Domínios de Homologia de src/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Células CACO-2 , Adesão Celular , Linhagem Celular , Polaridade Celular , Citoplasma/metabolismo , Análise Mutacional de DNA , Fibroblastos/metabolismo , Imunofluorescência , Células HeLa , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Células NIH 3T3 , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteínas de Ligação a RNA , Ratos , Transdução de Sinais , Domínios de Homologia de src/fisiologiaRESUMO
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.
Assuntos
NAD , Tiazinas , Morte Celular , NAD/metabolismo , Piperazinas , ProteômicaRESUMO
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.
Assuntos
Fibroblastos Associados a Câncer/efeitos dos fármacos , Carcinoma Ductal Pancreático/patologia , Células Endoteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Células Cultivadas , Citocina TWEAK/genética , Citocina TWEAK/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Masculino , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor de TIE-1/genética , Receptor de TIE-1/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/genética , Regulação da Expressão Gênica , Proteínas de Neoplasias/genética , Pâncreas/metabolismo , Resposta a Proteínas não Dobradas/genética , Células Acinares/metabolismo , Células Acinares/ultraestrutura , Animais , Western Blotting , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas de Neoplasias/metabolismo , Pâncreas/citologia , Pâncreas/ultraestrutura , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Adenocarcinoma/metabolismo , Resistencia a Medicamentos Antineoplásicos , Metaboloma , Metabolômica , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/etiologia , Adenocarcinoma/mortalidade , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biologia Computacional/métodos , Bases de Dados Genéticas , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Metabolômica/métodos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/mortalidade , FenótipoRESUMO
Pancreatic cancer is one of the worst, as almost 100% of patients will die within 5 years after diagnosis. The tumors are characterized by an early, invasive, and metastatic phenotype, and extreme resistance to all known anticancer therapies. Therefore, there is an urgent need to develop new investigative strategies in order to identify new molecular targets and, possibly, new drugs to fight this disease efficiently. Whereas it has been known for more than 3 decades now, ubiquitylation is a post-translational modification of protein that only recently emerged as a major regulator of many biological functions, dependent and independent on the proteasome, whose failure is involved in many human diseases, including cancer. Indeed, despite its role in promoting protein degradation through the proteasome, ubiquitylation is now known to regulate diverse cellular processes, such as membrane protein endocytosis and intracellular trafficking, assembly of protein complexes, gene transcription, and activation or inactivation of enzymes. Taking into account that ubiquitylation machinery is a three-step process involving hundreds of proteins, which is countered by numerous ubiquitin hydrolases, and that the function of ubiquitylation relies on the recognition of the ubiquitin signals by hundreds of proteins containing a ubiquitin binding domain (including the proteasome), the number of possible therapeutic targets is exceptionally vast and will need to be explored carefully for each disease. In the case of pancreatic cancer, the study and the identification of specific alteration(s) in protein ubiquitylation may help to explain its severity and may furnish more specific targets for more efficient therapies.
Assuntos
Neoplasias Pancreáticas/metabolismo , Proteínas/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Animais , Humanos , Modelos Biológicos , Neoplasias Pancreáticas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Carcinoma Ductal Pancreático/tratamento farmacológico , Núcleo Celular/metabolismo , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Piperazinas/farmacologia , Tiazinas/farmacologia , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Núcleo Celular/efeitos dos fármacos , Proliferação de Células , Reparo do DNA , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transporte Proteico , Sumoilação , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
RESUMO
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.
Assuntos
Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Linhagem Celular , Cromatografia Líquida , Humanos , Software , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Antineoplásicos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Necroptose/efeitos dos fármacos , Proteínas de Neoplasias , Neoplasias/tratamento farmacológico , Trifluoperazina , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Hep G2 , Humanos , Células Jurkat , Camundongos , Camundongos Nus , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Células PC-3 , Trifluoperazina/análogos & derivados , Trifluoperazina/síntese química , Trifluoperazina/química , Trifluoperazina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.