Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
1.
Proc Natl Acad Sci U S A ; 121(5): e2308776121, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38252831

RESUMO

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.


Assuntos
Influenza Humana , Vírus , Animais , Camundongos , Humanos , Proteoma , Peptídeos/farmacologia , Descoberta de Drogas
2.
Cell Death Dis ; 14(6): 392, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37391438

RESUMO

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.


Assuntos
Neoplasias da Mama , Leucemia , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Terapia Neoadjuvante , Polimorfismo de Nucleotídeo Único/genética , Retículo Endoplasmático , Biomarcadores
3.
iScience ; 26(5): 106674, 2023 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-37182099

RESUMO

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.

4.
EMBO Rep ; 24(4): e56524, 2023 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-36802267

RESUMO

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.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/genética , Pericitos , Proteínas Proto-Oncogênicas p21(ras) , Células-Tronco , Microambiente Tumoral , Neoplasias Pancreáticas
5.
J Exp Clin Cancer Res ; 41(1): 324, 2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36380366

RESUMO

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.


Assuntos
Neoplasias da Mama , Receptores ErbB , Humanos , Feminino , Espécies Reativas de Oxigênio , Receptores ErbB/metabolismo , Neoplasias da Mama/genética , Oxigênio/metabolismo , Hipóxia
6.
Biomater Sci ; 10(2): 485-498, 2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-34904143

RESUMO

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.


Assuntos
Medula Óssea , Osso e Ossos , Células da Medula Óssea , Matriz Extracelular , Humanos , Reprodutibilidade dos Testes
7.
Elife ; 102021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34415238

RESUMO

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.


Assuntos
Quimiotaxia , Dictyostelium/fisiologia , Oxigênio/metabolismo , Anaerobiose
9.
PLoS Pathog ; 17(2): e1009340, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33596274

RESUMO

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.


Assuntos
Antivirais/administração & dosagem , Retículo Endoplasmático/patologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem , Retículo Endoplasmático/virologia , Humanos , Imunidade Inata , Influenza Humana/patologia , Influenza Humana/virologia , Mitocôndrias/patologia , Mitocôndrias/virologia , Replicação Viral
10.
iScience ; 23(6): 101236, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32563156

RESUMO

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.

11.
Oncogene ; 39(15): 3056-3074, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32066881

RESUMO

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.


Assuntos
Neoplasias da Mama/patologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína bcl-X/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Metástase Linfática/patologia , Mitocôndrias/efeitos dos fármacos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Nitrofenóis/farmacologia , Nitrofenóis/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Canal de Ânion 1 Dependente de Voltagem/antagonistas & inibidores , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genética
12.
Nat Commun ; 9(1): 4545, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30382089

RESUMO

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.


Assuntos
Movimento Celular , Receptores ErbB/metabolismo , Glândulas Mamárias Humanas/citologia , Oxigênio/farmacologia , Hipóxia Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Oxirredução , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Cancer Res ; 78(6): 1404-1417, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29330143

RESUMO

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.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Retículo Endoplasmático/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/fisiologia , Sítios de Ligação , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos SCID , Terapia de Alvo Molecular/métodos , Fragmentos de Peptídeos/metabolismo , Peptídeos/farmacologia , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Biochim Biophys Acta Gene Regul Mech ; 1860(4): 427-437, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28215965

RESUMO

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.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Subunidades beta de Inibinas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Loci Gênicos , Subunidades beta de Inibinas/metabolismo , Lisina , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transdução de Sinais
15.
J Proteomics ; 110: 183-94, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25242195

RESUMO

Disease phenotype reorganizations are the consequences of signaling pathway perturbations and protein abundance modulations. Characterizing the protein signature of a biological event allows the identification of new candidate biomarkers, new targets for treatments and selective patient therapy. The combination of discovery LC-MS/MS analyses and targeted mass spectrometry using selected reaction monitoring (SRM) mode has emerged as a powerful technology for biomarker identification and quantification owing to faster development time and multiplexing capability. The epithelial-mesenchymal transition (EMT) is a process that controls local invasion and metastasis generation by stimulating changes in adhesion and migration of cells but also in metabolic pathways. In this study, the non-transformed human breast epithelial cell line MCF10A, treated by TGFß or overexpressing mutant K-Ras(v12), two EMT inducers frequently involved in cancer progression, was used to characterize protein abundance changes during an EMT event. The LC-MS/MS analysis and label-free quantification revealed that TGFß and K-Ras(v12) induce a similar pattern of protein regulation and that besides the expected cytoskeletal changes, a strong increase in the anabolism and energy production machinery was observed. BIOLOGICAL SIGNIFICANCE: To our knowledge, this is the first proteomic analysis combining a label-free quantification with an SRM validation of proteins regulated by TGFß and K-Rasv12. This study reveals new insights in the characterization of the changes occurring during an epithelial-mesenchymal transition (EMT) event. Notably, a strong increase in the anabolism and energy production machinery was observed upon both EMT inducers.


Assuntos
Mama/metabolismo , Cromatografia Líquida/métodos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Espectrometria de Massas/métodos , Fator de Crescimento Transformador beta/metabolismo , Proteínas ras/metabolismo , Linhagem Celular , Feminino , Humanos , Mapeamento de Peptídeos/métodos , Coloração e Rotulagem , Fator de Crescimento Transformador beta/química , Proteínas ras/química
16.
Cancer Res ; 73(22): 6621-31, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24078802

RESUMO

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process that converts epithelial cells into highly motile mesenchymal cells. This physiologic process occurs largely during embryonic development but is aberrantly reactivated in different pathologic situations, including fibrosis and cancer. We conducted a siRNA screening targeted to the human kinome with the aim of discovering new EMT effectors. With this approach, we have identified mTOR complex 1 (mTORC1), a nutrient sensor that controls protein and lipid synthesis, as a key regulator of epithelial integrity. Using a combination of RNAi and pharmacologic approaches, we report here that inhibition of either mTOR or RPTOR triggers EMT in mammary epithelial cells. This EMT was characterized by the induction of the mesenchymal markers such as fibronectin, vimentin, and PAI-1, together with the repression of epithelial markers such as E-cadherin and ZO-3. In addition, mTORC1 blockade enhanced in vivo migratory properties of mammary cells and induced EMT independent of the TGF-ß pathway. Finally, among the transcription factors known to activate EMT, both ZEB1 and ZEB2 were upregulated following mTOR repression. Their increased expression correlated with a marked reduction in miR-200b and miR-200c mRNA levels, two microRNAs known to downregulate ZEB1 and ZEB2 expression. Taken together, our findings unravel a novel function for mTORC1 in maintaining the epithelial phenotype and further indicate that this effect is mediated through the opposite regulation of ZEB1/ZEB2 and miR-200b and miR-200c. Furthermore, these results suggest a plausible etiologic explanation for the progressive pulmonary fibrosis, a frequent adverse condition associated with the therapeutic use of mTOR inhibitors.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Complexos Multiproteicos/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Embrião de Galinha , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina , MicroRNAs/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Invasividade Neoplásica , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Homeobox 1 de Ligação a E-box em Dedo de Zinco
17.
J Cell Sci ; 126(Pt 16): 3713-23, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23788427

RESUMO

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.


Assuntos
Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/fisiologia , Humanos , Dados de Sequência Molecular , Inibidor 1 de Ativador de Plasminogênio/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Sumoilação , Transfecção
18.
Prenat Diagn ; 33(10): 990-5, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23784867

RESUMO

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.


Assuntos
Líquido Amniótico/metabolismo , Biomarcadores/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Defeitos do Tubo Neural/diagnóstico , Acetilcolinesterase/análise , Acetilcolinesterase/metabolismo , Adulto , Líquido Amniótico/química , Biomarcadores/análise , Estudos de Casos e Controles , Feminino , Idade Gestacional , Proteína Glial Fibrilar Ácida/análise , Humanos , Defeitos do Tubo Neural/metabolismo , Gravidez , Adulto Jovem , alfa-Fetoproteínas/análise , alfa-Fetoproteínas/metabolismo
19.
Mol Cell Proteomics ; 12(7): 1939-52, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23579184

RESUMO

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.


Assuntos
Aptâmeros de Peptídeos/metabolismo , Calcineurina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Aptâmeros de Peptídeos/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Ratos , Técnicas do Sistema de Duplo-Híbrido
20.
Development ; 140(1): 167-75, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23154409

RESUMO

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.


Assuntos
Diferenciação Celular/genética , Células Epiteliais/citologia , Lactação/genética , Glândulas Mamárias Animais/citologia , Proteína Smad4/antagonistas & inibidores , Fatores de Transcrição/fisiologia , Animais , Células Epiteliais/fisiologia , Feminino , Masculino , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Transdução de Sinais/genética , Proteína Smad4/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...