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We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.
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Gripe Humana , Virus , Animales , Ratones , Humanos , Proteoma , Péptidos/farmacología , Descubrimiento de DrogasRESUMEN
We report the identification of a cell population that shares pericyte, stromal and stemness features, does not harbor the KrasG12D mutation and drives tumoral growth in vitro and in vivo. We term these cells pericyte stem cells (PeSCs) and define them as CD45- EPCAM- CD29+ CD106+ CD24+ CD44+ cells. We perform studies with p48-Cre;KrasG12D (KC), pdx1-Cre;KrasG12D ;Ink4a/Arffl/fl (KIC) and pdx1-Cre;KrasG12D ;p53R172H (KPC) and tumor tissues from PDAC and chronic pancreatitis patients. We also perform single-cell RNAseq analysis and reveal a unique signature of PeSC. Under steady-state conditions, PeSCs are barely detectable in the pancreas but present in the neoplastic microenvironment both in humans and mice. The coinjection of PeSCs and tumor epithelial cells leads to increased tumor growth, differentiation of Ly6G+ myeloid-derived suppressor cells, and a decreased amount of F4/80+ macrophages and CD11c+ dendritic cells. This population induces resistance to anti-PD-1 immunotherapy when coinjected with epithelial tumor cells. Our data reveal the existence of a cell population that instructs immunosuppressive myeloid cell responses to bypass PD-1 targeting and thus suggest potential new approaches for overcoming resistance to immunotherapy in clinical settings.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/genética , Pericitos , Proteínas Proto-Oncogénicas p21(ras) , Células Madre , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Influenza virus infections are major public health threats due to their high rates of morbidity and mortality. Upon influenza virus entry, host cells experience modifications of endomembranes, including those used for virus trafficking and replication. Here we report that influenza virus infection modifies mitochondrial morphodynamics by promoting mitochondria elongation and altering endoplasmic reticulum-mitochondria tethering in host cells. Expression of the viral RNA recapitulates these modifications inside cells. Virus induced mitochondria hyper-elongation was promoted by fission associated protein DRP1 relocalization to the cytosol, enhancing a pro-fusion status. We show that altering mitochondrial hyper-fusion with Mito-C, a novel pro-fission compound, not only restores mitochondrial morphodynamics and endoplasmic reticulum-mitochondria contact sites but also dramatically reduces influenza replication. Finally, we demonstrate that the observed Mito-C antiviral property is directly connected with the innate immunity signaling RIG-I complex at mitochondria. Our data highlight the importance of a functional interchange between mitochondrial morphodynamics and innate immunity machineries in the context of influenza viral infection.
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Antivirales/administración & dosificación , Retículo Endoplásmico/patología , Interacciones Huésped-Patógeno , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Preparaciones Farmacéuticas/administración & dosificación , Retículo Endoplásmico/virología , Humanos , Inmunidad Innata , Gripe Humana/patología , Gripe Humana/virología , Mitocondrias/patología , Mitocondrias/virología , Replicación ViralRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009340.].
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Transforming growth factor ß (TGFß) is widely recognised as an important factor that regulates many steps of normal mammary gland (MG) development, including branching morphogenesis, functional differentiation and involution. Tif1γ has previously been reported to temporally and spatially control TGFß signalling during early vertebrate development by exerting negative effects over SMAD4 availability. To evaluate the contribution of Tif1 γ to MG development, we developed a Cre/LoxP system to specifically invalidate the Tif1g gene in mammary epithelial cells in vivo. Tif1g-null mammary gland development appeared to be normal and no defects were observed during the lifespan of virgin mice. However, a lactation defect was observed in mammary glands of Tif1g-null mice. We demonstrate that Tif1 γ is essential for the terminal differentiation of alveolar epithelial cells at the end of pregnancy and to ensure lactation. Tif1 γ appears to play a crucial role in the crosstalk between TGFß and prolactin pathways by negatively regulating both PRL receptor expression and STAT5 phosphorylation, thereby impairing the subsequent transactivation of PRL target genes. Using HC11 cells as a model, we demonstrate that the effects of Tif1g knockdown on lactation depend on both SMAD4 and TGFß. Interestingly, we found that the Tif1γ expression pattern in mammary epithelial cells is almost symmetrically opposite to that described for TGFß. We propose that Tif1γ contributes to the repression of TGFß activity during late pregnancy and prevents lactation by inhibiting SMAD4.
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Diferenciación Celular/genética , Células Epiteliales/citología , Lactancia/genética , Glándulas Mamarias Animales/citología , Proteína Smad4/antagonistas & inhibidores , Factores de Transcripción/fisiología , Animales , Células Epiteliales/fisiología , Femenino , Masculino , Glándulas Mamarias Animales/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Embarazo , Transducción de Señal/genética , Proteína Smad4/fisiologíaRESUMEN
TIF1γ, a new regulator of TGFß signaling, inhibits the Smad4-mediated TGFß response by interaction with Smad2/3 or ubiquitylation of Smad4. We have shown that TIF1γ participates in TGFß signaling as a negative regulator of Smad4 during the TGFß-induced epithelial-to-mesenchymal transition (EMT) in mammary epithelial cells, and during terminal differentiation of mammary alveolar epithelial cells and lactation. We demonstrate here that TIF1γ is sumoylated and interacts with Ubc9, the only known SUMO-conjugating enzyme. Four functional sumoylation sites lie within the middle domain of TIF1γ, the Smad interaction domain. We show that a sumoylation-defective TIF1γ mutant significantly reduces TIF1γ inhibition of Smad complexes and that of the Smad-mediated TGFß transcriptional response. Moreover, chromatin immunoprecipitation experiments indicate that TIF1γ sumoylation is required to limit Smad4 binding on the PAI-1 TGFß target gene promoter. Ectopic expression of TIF1γ in mammary epithelial cells inhibits TGFß-induced EMT, an effect relieved by expression of non-sumoylated TIF1γ. Taken together, our results identify a new TGFß regulatory layer, whereby sumoylation strengthens the TIF1γ repressive action on canonical TGFß signaling.
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Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular/fisiología , Humanos , Datos de Secuencia Molecular , Inhibidor 1 de Activador Plasminogénico/genética , Regiones Promotoras Genéticas , Transducción de Señal , Proteínas Smad/genética , Proteínas Smad/metabolismo , Sumoilación , TransfecciónRESUMEN
We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two binding proteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2 and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.
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Aptámeros de Péptidos/metabolismo , Calcineurina/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Aptámeros de Péptidos/genética , Línea Celular Tumoral , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Ratas , Técnicas del Sistema de Dos HíbridosRESUMEN
Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localisation and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localisation is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we demonstrated that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs displayed reduced 1,4,5-inositol trisphosphate receptor (IP3R)-mediated ER calcium release downstream of Phospholipase C activation. Collectively, our data indicate that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the level of the ER, in addition to its well-known role in regulating apoptosis through the mitochondria.
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TGF-ß is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-ß signalling. To understand the role of TIF1γ in TGF-ß signalling and its requirement for EMT, we analysed the TGF-ß1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-ß1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-ß signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-ß-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-ß signalling and EMT.
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Células Epiteliales/metabolismo , Glándulas Mamarias Humanas/metabolismo , Proteína Smad4/metabolismo , Factores de Transcripción/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Epiteliales/citología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Humanos , Glándulas Mamarias Humanas/citología , Proteína Smad4/genética , Factores de Transcripción/genética , Factores de Transcripción/farmacología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
OBJECTIVE: Neural tube defects (NTDs) are usually identified by ultrasonography and confirmed by alpha-fetoprotein (AFP) assay and acetylcholinesterase (AchE) electrophoresis in amniotic fluid. Yet, both of these biomarkers can be found positive in other etiologies. Here, amniotic fluid glial fibrillary acidic protein (AF-GFAP), which was identified by a proteomic study, is shown to be a useful biomarker for NTD diagnosis. METHOD: Amniotic fluid glial fibrillary acidic protein was measured by an ELISA assay in 138 cases of NTDs. Seventy samples from normal pregnancies used as controls and 27 samples giving false positive or false negative results either for AchE or AFP and corresponding to fetal death (n = 8), gastroschisis (n = 8), and unexplained etiologies (n = 11) were also tested. RESULTS: Whatever the gestational age, GFAP was below 0.2 ng/mL in control samples, whereas 99.1% of open NTDs (29/29 in the anterior NTD group and 80/81 in the spina bifida group) were above this threshold. Closed NTDs were all negative (28/28). None of the other samples tested were positive, except in case of fetal death (8/8). CONCLUSIONS: Amniotic fluid glial fibrillary acidic protein is a sensitive biomarker for open NTD diagnosis with a good negative predictive value for closed NTD. Compared with AFP and AchE, our results indicate that AF-GFAP alone is more efficient than this classical association.
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Líquido Amniótico/metabolismo , Biomarcadores/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Defectos del Tubo Neural/diagnóstico , Acetilcolinesterasa/análisis , Acetilcolinesterasa/metabolismo , Adulto , Líquido Amniótico/química , Biomarcadores/análisis , Estudios de Casos y Controles , Femenino , Edad Gestacional , Proteína Ácida Fibrilar de la Glía/análisis , Humanos , Defectos del Tubo Neural/metabolismo , Embarazo , Adulto Joven , alfa-Fetoproteínas/análisis , alfa-Fetoproteínas/metabolismoRESUMEN
Overexpression of Bcl-2 proteins such as Bcl2L10, also referred to as Nrh, is associated with resistance to therapy and poor survival in various cancers, including breast cancer, lung cancer, and leukemia. The single nucleotide polymorphism (SNP) of BCL2L10 in its BH4 domain at position 11 (BCL2L10 Leu11Arg, rs2231292), corresponding to position 11 in the Nrh open reading frame, is reported to lower resistance towards chemotherapy, with patients showing better survival in the context of acute leukemia and colorectal cancer. Using cellular models and clinical data, we aimed to extend this knowledge to breast cancer. We report that the homozygous status of the Nrh Leu11Arg isoform (Nrh-R) is found in 9.7-11% percent of the clinical datasets studied. Furthermore, Nrh-R confers higher sensitivity towards Thapsigargin-induced cell death compared to the Nrh-L isoform, due to altered interactions with IP3R1 Ca2+ channels in the former case. Collectively, our data show that cells expressing the Nrh-R isoform are more prone to death triggered by Ca2+ stress inducers, compared to Nrh-L expressing cells. Analysis of breast cancer cohorts revealed that patients genotyped as Nrh-R/Nrh-R may have a better outcome. Overall, this study supports the notion that the rs2231292 Nrh SNP could be used as a predictive tool regarding chemoresistance, improving therapeutic decision-making processes. Moreover, it sheds new light on the contribution of the BH4 domain to the anti-apoptotic function of Nrh and identifies the IP3R1/Nrh complex as a potential therapeutic target in the context of breast cancer.
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Neoplasias de la Mama , Leucemia , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Terapia Neoadyuvante , Polimorfismo de Nucleótido Simple/genética , Retículo Endoplásmico , BiomarcadoresRESUMEN
Non-lethal caspase activation (NLCA) has been linked to neurodevelopmental processes. However, how neurons control NLCA remains elusive. Here, we focused on Bcl-xL, a Bcl-2 homolog regulating caspase activation through the mitochondria. We generated a mouse model, referred to as ER-xL, in which Bcl-xL is absent in the mitochondria, yet present in the endoplasmic reticulum. Unlike bclx knockout mice that died at E13.5, ER-xL mice survived embryonic development but died post-partum because of altered feeding behavior. Enhanced caspase-3 activity was observed in the brain and the spinal cord white matter, but not the gray matter. No increase in cell death was observed in ER-xL cortical neurons, suggesting that the observed caspase-3 activation was apoptosis-independent. ER-xL neurons displayed increased caspase-3 activity in the neurites, resulting in impaired axon arborescence and synaptogenesis. Together, our findings suggest that mitochondrial Bcl-xL finely tunes caspase-3 through Drp-1-dependent mitochondrial fission, which is critical to neural network design.
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BACKGROUND: Aerotaxis, the chemotactism to oxygen, is well documented in prokaryotes. We previously reported for the first time that non-tumorigenic breast epithelial cells also display unequivocal directional migration towards oxygen. This process is independent of the hypoxia-inducible factor (HIF)/prolyl hydroxylase domain (PHD) pathway but controlled by the redox regulation of epidermal growth factor receptor (EGFR), with a reactive oxygen species (ROS) gradient overlapping the oxygen gradient at low oxygen concentration. Since hypoxia is an acknowledged hallmark of cancers, we addressed the putative contribution of aerotaxis to cancer metastasis by studying the directed migration of cancer cells from an hypoxic environment towards nearby oxygen sources, modelling the in vivo migration of cancer cells towards blood capillaries. METHODS: We subjected to the aerotactic test described in our previous papers cells isolated from fresh breast tumours analysed by the Pathology Department of the Saint-Etienne University Hospital (France) over a year. The main selection criterion, aside from patient consent, was the size of the tumour, which had to be large enough to perform the aerotactic tests without compromising routine diagnostic tests. Finally, we compared the aerotactic properties of these primary cells with those of commonly available breast cancer cell lines. RESULTS: We show that cells freshly isolated from sixteen human breast tumour biopsies, representative of various histological characteristics and grades, are endowed with strong aerotactic properties similar to normal mammary epithelial cell lines. Strikingly, aerotaxis of these primary cancerous cells is also strongly dependent on both EGFR activation and ROS. In addition, we demonstrate that aerotaxis can trigger directional invasion of tumour cells within the extracellular matrix contrary to normal mammary epithelial cells. This contrasts with results obtained with breast cancer cell lines, in which aerotactic properties were either retained or impaired, and in some cases, even lost during the establishment of these cell lines. CONCLUSIONS: Altogether, our results support that aerotaxis may play an important role in breast tumour metastasis. In view of these findings, we discuss the prospects for combating metastatic spread. TRIAL REGISTRATION: IRBN1462021/CHUSTE.
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Neoplasias de la Mama , Receptores ErbB , Humanos , Femenino , Especies Reactivas de Oxígeno , Receptores ErbB/metabolismo , Neoplasias de la Mama/genética , Oxígeno/metabolismo , HipoxiaRESUMEN
Bone marrow is a complex and dynamic microenvironment that provides essential cues to resident cells. We developed a standardized three-dimensional (3D) model to decipher mechanisms that control human cells during hematological and non-hematological processes. Our simple 3D-model is constituted of a biphasic calcium phosphate-based scaffold and human cell lines to ensure a high reproducibility. We obtained a minimal well-organized bone marrow-like structure in which various cell types and secreted extracellular matrix can be observed and characterized by in situ imaging or following viable cell retrieval. The complexity of the system can be increased and customized, with each cellular component being independently modulated according to the issue investigated. Introduction of pathological elements in this 3D-system accurately reproduced changes observed in patient bone marrow. Hence, we have developed a handy and flexible standardized microphysiological system that mimics human bone marrow, allowing histological analysis and functional assays on collected cells.
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Médula Ósea , Huesos , Células de la Médula Ósea , Matriz Extracelular , Humanos , Reproducibilidad de los ResultadosRESUMEN
Using a self-generated hypoxic assay, we show that the amoeba Dictyostelium discoideum displays a remarkable collective aerotactic behavior. When a cell colony is covered, cells quickly consume the available oxygen (O2) and form a dense ring moving outwards at constant speed and density. To decipher this collective process, we combined two technological developments: porphyrin-based O2 -sensing films and microfluidic O2 gradient generators. We showed that Dictyostelium cells exhibit aerotactic and aerokinetic response in a low range of O2 concentration indicative of a very efficient detection mechanism. Cell behaviors under self-generated or imposed O2 gradients were modeled using an in silico cellular Potts model built on experimental observations. This computational model was complemented with a parsimonious 'Go or Grow' partial differential equation (PDE) model. In both models, we found that the collective migration of a dense ring can be explained by the interplay between cell division and the modulation of aerotaxis.
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Quimiotaxis , Dictyostelium/fisiología , Oxígeno/metabolismo , AnaerobiosisRESUMEN
The progesterone receptor (PR) is an inducible transcription factor that plays critical roles in female reproductive processes and in several aspects of breast cancer tumorigenesis. Our report describes the type I protein arginine methyltransferase 1 (PRMT1) as a cofactor controlling progesterone pathway, through the direct methylation of PR. Mechanistic assays in breast cancer cells indicate that PRMT1 methylates PR at the arginine 637 and reduces the stability of the receptor, thereby accelerating its recycling and finally its transcriptional activity. Depletion of PRMT1 decreases the expression of a subset of progesterone-inducible genes, controlling breast cancer cells proliferation and migration. Consistently, Kaplan-Meier analysis revealed that low expression of PRMT1 predicts a longer survival among the subgroup with high PR. Our study highlights PR methylation as a molecular switch adapting the transcription requirement of breast cells during tumorigenesis.
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The Bcl-xL apoptosis inhibitor plays a major role in vertebrate development. In addition to its effect on apoptosis, Bcl-xL is also involved in cell migration and mitochondrial metabolism. These effects may favour the onset and dissemination of metastasis. However, the underlying molecular mechanisms remain to be fully understood. Here we focus on the control of cell migration by Bcl-xL in the context of breast cancer cells. We show that Bcl-xL silencing led to migration defects in Hs578T and MDA-MB231 cells. These defects were rescued by re-expressing mitochondria-addressed, but not endoplasmic reticulum-addressed, Bcl-xL. The use of BH3 mimetics, such as ABT-737 and WEHI-539 confirmed that the effect of Bcl-xL on migration did not depend on interactions with BH3-containing death accelerators such as Bax or BH3-only proteins. In contrast, the use of a BH4 peptide that disrupts the Bcl-xL/VDAC1 complex supports that Bcl-xL by acting on VDAC1 permeability contributes to cell migration through the promotion of reactive oxygen species production by the electron transport chain. Collectively our data highlight the key role of Bcl-xL at the interface between cell metabolism, cell death, and cell migration, thus exposing the VDAC1/Bcl-xL interaction as a promising target for anti-tumour therapy in the context of metastatic breast cancer.
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Neoplasias de la Mama/patología , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína bcl-X/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/uso terapéutico , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Compuestos de Bifenilo/farmacología , Compuestos de Bifenilo/uso terapéutico , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Metástasis Linfática/patología , Mitocondrias/efectos de los fármacos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Invasividad Neoplásica/prevención & control , Nitrofenoles/farmacología , Nitrofenoles/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Unión Proteica/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Canal Aniónico 1 Dependiente del Voltaje/antagonistas & inhibidores , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/genéticaRESUMEN
Aerotaxis or chemotaxis to oxygen was described in bacteria 130 years ago. In eukaryotes, the main adaptation to hypoxia currently described relies on HIF transcription factors. To investigate whether aerotaxis is conserved in higher eukaryotes, an approach based on the self-generation of hypoxia after cell confinement was developed. We show that epithelial cells from various tissues migrate with an extreme directionality towards oxygen to escape hypoxia, independently of the HIF pathway. We provide evidence that, concomitant to the oxygen gradient, a gradient of reactive oxygen species (ROS) develops under confinement and that antioxidants dampen aerotaxis. Finally, we establish that in mammary cells, EGF receptor, the activity of which is potentiated by ROS and inhibited by hypoxia, represents the molecular target that guides hypoxic cells to oxygen. Our results reveals that aerotaxis is a property of higher eukaryotic cells and proceeds from the conversion of oxygen into ROS.
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Movimiento Celular , Receptores ErbB/metabolismo , Glándulas Mamarias Humanas/citología , Oxígeno/farmacología , Hipoxia de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Oxidación-Reducción , Procolágeno-Prolina Dioxigenasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Drug resistance and metastatic relapse remain a top challenge in breast cancer treatment. In this study, we present preclinical evidence for a strategy to eradicate advanced breast cancers by targeting the BCL-2 homolog Nrh/BCL2L10, which we discovered to be overexpressed in >45% of a large cohort of breast invasive carcinomas. Nrh expression in these tumors correlated with reduced metastasis-free survival, and we determined it to be an independent marker of poor prognosis. Nrh protein localized to the endoplasmic reticulum. Mechanistic investigations showed that Nrh made BH4 domain-dependent interactions with the ligand-binding domain of the inositol-1,4,5-triphosphate receptor (IP3R), a type 1/3 Ca2+ channel, allowing Nrh to negatively regulate ER-Ca2+ release and to mediate antiapoptosis. Notably, disrupting Nrh/IP3R complexes by BH4 mimetic peptides was sufficient to inhibit the growth of breast cancer cells in vitro and in vivo Taken together, our results highlighted Nrh as a novel prognostic marker and a candidate therapeutic target for late stage breast cancers that may be addicted to Nrh.Significance: These findings offer a comprehensive molecular model for the activity of Nrh/BCL2L10, a little studied antiapoptotic molecule, prognostic marker, and candidate drug target in breast cancer. Cancer Res; 78(6); 1404-17. ©2018 AACR.
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Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Retículo Endoplásmico/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/fisiología , Sitios de Unión , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Calcio/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Retículo Endoplásmico/efectos de los fármacos , Femenino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones SCID , Terapia Molecular Dirigida/métodos , Fragmentos de Péptidos/metabolismo , Péptidos/farmacología , Pronóstico , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although Men1 is a well-known tumour suppressor gene, little is known about the functions of Menin, the protein it encodes for. Since few years, numerous publications support a major role of Menin in the control of epigenetics gene regulation. While Menin interaction with MLL complex favours transcriptional activation of target genes through H3K4me3 marks, Menin also represses gene expression via mechanisms involving the Polycomb repressing complex (PRC). Interestingly, Ezh2, the PRC-methyltransferase that catalyses H3K27me3 repressive marks and Menin have been shown to co-occupy a large number of promoters. However, lack of binding between Menin and Ezh2 suggests that another member of the PRC complex is mediating this indirect interaction. Having found that ActivinB - a TGFß superfamily member encoded by the Inhbb gene - is upregulated in insulinoma tumours caused by Men1 invalidation, we hypothesize that Menin could directly participate in the epigenetic-repression of Inhbb gene expression. Using Animal model and cell lines, we report that loss of Menin is directly associated with ActivinB-induced expression both in vivo and in vitro. Our work further reveals that ActivinB expression is mediated through a direct modulation of H3K27me3 marks on the Inhbb locus in Menin-KO cell lines. More importantly, we show that Menin binds on the promoter of Inhbb gene where it favours the recruitment of Ezh2 via an indirect mechanism involving Akt-phosphorylation. Our data suggests therefore that Menin could take an important part to the Ezh2-epigenetic repressive landscape in many cells and tissues through its capacity to modulate Akt phosphorylation.