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1.
EMBO J ; 40(3): e104895, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33320356

RESUMO

The Hippo signaling pathway is a major regulator of organ growth, which controls the activity of the transcription coactivator Yorkie (Yki) in Drosophila and its homolog YAP in mammals. Both Yki and YAP proteins exist as alternatively spliced isoforms containing either one or two WW domains. The biological importance of this conserved alternative splicing event is unknown. Here, we identify the splicing factor B52 as a regulator of yki alternative splicing in Drosophila and show that B52 modulates growth in part through modulation of yki alternative splicing. Yki isoforms differ by their transcriptional activity as well as their ability to bind and bridge PPxY motifs-containing partners, and can compete in vivo. Strikingly, flies in which yki alternative splicing has been abrogated, thus expressing only Yki2 isoform, exhibit fluctuating wing asymmetry, a signal of developmental instability. Our results identify yki alternative splicing as a new level of modulation of the Hippo pathway, that is required for growth equilibration during development. This study provides the first demonstration that the process of alternative splicing contributes to developmental robustness.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Processamento de RNA/metabolismo , Transativadores/genética , Transativadores/metabolismo , Processamento Alternativo , Animais , Linhagem Celular , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas Nucleares/química , Ligação Proteica , Domínios Proteicos , Fatores de Processamento de RNA/genética , Análise de Sequência de RNA , Transativadores/química , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo , Proteínas de Sinalização YAP
2.
Nucleic Acids Res ; 51(16): 8413-8433, 2023 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-37462077

RESUMO

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.


Assuntos
Daunorrubicina , Leucemia Mieloide Aguda , Humanos , Daunorrubicina/farmacologia , Daunorrubicina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Ésteres/uso terapêutico , Cromatina/genética
3.
Haematologica ; 109(1): 98-114, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37608777

RESUMO

Acute myeloid leukemias (AML) are severe hematomalignancies with dismal prognosis. The post-translational modification SUMOylation plays key roles in leukemogenesis and AML response to therapies. Here, we show that TAK-981 (subasumstat), a first-in-class SUMOylation inhibitor, is endowed with potent anti-leukemic activity in various preclinical models of AML. TAK-981 targets AML cell lines and patient blast cells in vitro and in vivo in xenografted mice with minimal toxicity on normal hematopoietic cells. Moreover, it synergizes with 5-azacytidine (AZA), a DNA-hypomethylating agent now used in combination with the BCL-2 inhibitor venetoclax to treat AML patients unfit for standard chemotherapies. Interestingly, TAK-981+AZA combination shows higher anti-leukemic activity than AZA+venetoclax combination both in vitro and in vivo, at least in the models tested. Mechanistically, TAK-981 potentiates the transcriptional reprogramming induced by AZA, promoting apoptosis, alteration of the cell cycle and differentiation of the leukemic cells. In addition, TAK-981+AZA treatment induces many genes linked to inflammation and immune response pathways. In particular, this leads to the secretion of type-I interferon by AML cells. Finally, TAK-981+AZA induces the expression of natural killer-activating ligands (MICA/B) and adhesion proteins (ICAM-1) at the surface of AML cells. Consistently, TAK-981+AZA-treated AML cells activate natural killer cells and increase their cytotoxic activity. Targeting SUMOylation with TAK-981 may thus be a promising strategy to both sensitize AML cells to AZA and reduce their immune-escape capacities.


Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Animais , Camundongos , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Sumoilação , Leucemia Mieloide Aguda/genética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Antineoplásicos/uso terapêutico
4.
Haematologica ; 107(11): 2562-2575, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-35172562

RESUMO

Resistance to chemotherapeutic drugs is a major cause of treatment failure in acute myeloid leukemias (AML). To better characterize the mechanisms of chemoresistance, we first identified genes whose expression is dysregulated in AML cells resistant to daunorubicin or cytarabine, the main drugs used for induction therapy. The genes found to be activated are mostly linked to immune signaling and inflammation. Among them, we identified a strong upregulation of the NOX2 NAPDH oxidase subunit genes (CYBB, CYBA, NCF1, NCF2, NCF4 and RAC2). The ensuing increase in NADPH oxidase expression and production of reactive oxygen species, which is particularly strong in daunorubicin-resistant cells, participates in the acquisition and/or maintenance of resistance to daunorubicin. Gp91phox (CYBB-encoded Nox2 catalytic subunit), was found to be more expressed and active in leukemic cells from patients with the French-American-British (FAB) M4/M5 subtypes of AML than in those from patients with the FAB M0-M2 ones. Moreover, its expression was increased at the surface of patients' chemotherapy-resistant AML cells. Finally, using a gene expression based score we demonstrated that high expression of NOX2 subunit genes is a marker of adverse prognosis in AML patients. The prognostic NOX score we defined is independent of the cytogenetic-based risk classification, FAB subtype, FLT3/NPM1 mutational status and age.


Assuntos
Leucemia Mieloide Aguda , NADPH Oxidase 2 , Humanos , Daunorrubicina , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/genética , Prognóstico , NADPH Oxidase 2/genética
5.
J Neurochem ; 139(3): 349-368, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27513819

RESUMO

Neuronal granules play an important role in the localization and transport of translationally silenced messenger ribonucleoproteins in neurons. Among the factors associated with these granules, the RNA-binding protein G3BP1 (stress-granules assembly factor) is involved in neuronal plasticity and is induced in Alzheimer's disease. We immunopurified a stable complex containing G3BP1 from mouse brain and performed high-throughput sequencing and cross-linking immunoprecipitation to identify the associated RNAs. The G3BP-complex contained the deubiquitinating protease USP10, CtBP1 and the RNA-binding proteins Caprin-1, G3BP2a and splicing factor proline and glutamine rich, or PSF. The G3BP-complex binds preferentially to transcripts that retain introns, and to non-coding sequences like 3'-untranslated region and long non-coding RNAs. Specific transcripts with retained introns appear to be enriched in the cerebellum compared to the rest of the brain and G3BP1 depletion decreased this intron retention in the cerebellum of G3BP1 knockout mice. Among the enriched transcripts, we found an overrepresentation of genes involved in synaptic transmission, especially glutamate-related neuronal transmission. Notably, G3BP1 seems to repress the expression of the mature Grm5 (metabotropic glutamate receptor 5) transcript, by promoting the retention of an intron in the immature transcript in the cerebellum. Our results suggest that G3BP is involved in a new functional mechanism to regulate non-coding RNAs including intron-retaining transcripts, and thus have broad implications for neuronal gene regulation, where intron retention is widespread.


Assuntos
Química Encefálica/genética , Proteínas de Transporte/metabolismo , Cerebelo/metabolismo , Íntrons/genética , Regiões 3' não Traduzidas/genética , Oxirredutases do Álcool/metabolismo , Animais , Reagentes de Ligações Cruzadas , Grânulos Citoplasmáticos/metabolismo , DNA Helicases , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas de Ligação a Poli-ADP-Ribose , RNA/biossíntese , RNA/genética , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA Longo não Codificante/genética , Transcrição Gênica , Ubiquitina Tiolesterase/metabolismo
6.
EMBO Rep ; 15(5): 529-39, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24639560

RESUMO

Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals.


Assuntos
Tecido Adiposo/fisiologia , Metabolismo Energético/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adipócitos/citologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Envelhecimento , Processamento Alternativo , Animais , Células Cultivadas , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lamina Tipo A/biossíntese , Longevidade/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias , Proteínas Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Progéria/genética , Isoformas de Proteínas , Precursores de Proteínas/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo
7.
J Immunother Cancer ; 12(7)2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-39029925

RESUMO

BACKGROUND: Natural killer (NK) cell therapy is considered an attractive and safe strategy for anticancer therapy. Nevertheless, when autologous or allogenic NK cells are used alone, the clinical benefit has been disappointing. This is partially due to the lack of target specificity. Recently, CD19-specific chimeric antigen receptor (CAR)-NK cells have proven to be safe and potent in patients with B-cell tumors. However, the generation of CAR-NK cells is a complicated manufacturing process. We aim at developing a targeted NK cell therapy without the need for cellular genetic modifications. We took advantage of the natural expression of the IgG Fc receptor CD16a (FcγRIIIa) to induce strong antigen-specific effector functions through antibody-dependent cell-mediated cytotoxicity (ADCC). We have generated the new technology "Pin", which enables the arming of modified monoclonal antibodies (mAbs) onto the CD16a of ex vivo expanded NK (eNK) cells. Methods Ex vivo eNK were prepared from umbilical cord blood cells and expanded using interleukin (IL)-2/IL-15 and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid feeder cells. mAbs were engineered with four substitutions called Pin mutations to increase their affinity to CD16a. eNK were incubated with anti-CD20 or anti-CD19 Pin-mAbs to generate "armed" eNK and were used to assess effector functions in vitro on cancer cell lines, lymphoma patient cells and in vivo. RESULTS: CD16a/Pin-mAb interaction is stable for several days and Pin-mAb eNK inherit the mAb specificity and exclusively induce ADCC against targets expressing the cognate antigen. Hence, Pin-mAbs confer long-term selectivity to eNK, which allows specific elimination of the target cells in several in vivo mouse models. Finally, we showed that it is possible to arm eNK with at least two Pin-mAbs simultaneously, to increase efficacy against heterogenous cancer cell populations. CONCLUSIONS: The Pin technology provides an off-the-shelf NK cell therapy platform to generate CAR-like NK cells, without genetic modifications, that easily target multiple tumor antigens.


Assuntos
Células Matadoras Naturais , Receptores de IgG , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Humanos , Animais , Camundongos , Receptores de IgG/metabolismo , Receptores de IgG/imunologia , Imunoterapia Adotiva/métodos , Linhagem Celular Tumoral , Antígenos CD19/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/farmacologia
8.
Hum Mol Genet ; 20(23): 4540-55, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21875900

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. Most cases of HGPS are due to a heterozygous silent mutation (c.1824C>T; p.Gly608Gly) that enhances the use of an internal 5' splice site (5'SS) in exon 11 of the LMNA pre-mRNA and leads to the production of a truncated protein (progerin) with a dominant negative effect. Here we show that HGPS mutation changes the accessibility of the 5'SS of LMNA exon 11 which is sequestered in a conserved RNA structure. Our results also reveal a regulatory role of a subset of serine-arginine (SR)-rich proteins, including serine-arginine rich splicing factor 1 (SRSF1) and SRSF6, on utilization of the 5'SS leading to lamin A or progerin production and a modulation of this regulation in the presence of the c.1824C>T mutation is shown directly on HGPS patient cells. Mutant mice carrying the equivalent mutation in the LMNA gene (c.1827C>T) also accumulate progerin and phenocopy the main cellular alterations and clinical defects of HGPS patients. RNAi-induced depletion of SRSF1 in the HGPS-like mouse embryonic fibroblasts (MEFs) allowed progerin reduction and dysmorphic nuclei phenotype correction, whereas SRSF6 depletion aggravated the HGPS-like MEF's phenotype. We demonstrate that changes in the splicing ratio between lamin A and progerin are key factors for lifespan since heterozygous mice harboring the mutation lived longer than homozygous littermates but less than the wild-type. Genetic and biochemical data together favor the view that physiological progerin production is under tight control of a conserved splicing mechanism to avoid precocious aging.


Assuntos
Senilidade Prematura/genética , Evolução Molecular , Lamina Tipo A/genética , Splicing de RNA/genética , Animais , Sequência de Bases , Células Cultivadas , Sequência Conservada/genética , Éxons/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Progéria/genética , Progéria/patologia , Isoformas de Proteínas/genética , Precursores de Proteínas/genética , RNA/química , RNA/genética , Sítios de Splice de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Processamento de Serina-Arginina , Transfecção
9.
J Cell Biol ; 178(1): 23-30, 2007 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-17606864

RESUMO

In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA-ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis.


Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Forma Celular/fisiologia , Células Cultivadas , Colágeno/metabolismo , Combinação de Medicamentos , Eletroporação , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Laminina/metabolismo , Camundongos , Proteoglicanas/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/análise
10.
J Control Release ; 341: 578-590, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34915070

RESUMO

Monoclonal antibodies (mAbs) are large size molecules that have demonstrated high therapeutic potential for the treatment of cancer or autoimmune diseases. Despite some excellent results, their intravenous administration results in high plasma concentration. This triggers off-target effects and sometimes poor targeted tissue distribution. To circumvent this issue, we investigated a local controlled-delivery approach using an in situ forming depot technology. Two clinically relevant mAbs, rituximab (RTX) and daratumumab (DARA), were formulated using an injectable technology based on biodegradable PEG-PLA copolymers. The stability and controlled release features of the formulations were investigated. HPLC and mass spectrometry revealed the preservation of the protein structure. In vitro binding of formulated antibodies to their target antigens and to their cellular FcγRIIIa natural killer cell receptor was fully maintained. Furthermore, encapsulated RTX was as efficient as classical intravenous RTX treatment to inhibit the in vivo tumor growth of malignant human B cells in immunodeficient NSG mice. Finally, the intra-articular administration of the formulated mAbs yielded a sustained local release associated with a lower plasma concentration compared to the intra-articular delivery of non-encapsulated mAbs. Our results demonstrate that the utilization of this polymeric technology is a reliable alternative for the local delivery of fully functional clinically relevant mAbs.


Assuntos
Polímeros , Animais , Preparações de Ação Retardada/química , Camundongos , Polímeros/química
11.
RNA Biol ; 7(4): 495-503, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20864806

RESUMO

Alternative splicing is a key molecular mechanism for increasing the complexity of the human transcriptome. Nearly all human genes are regulated by alternative splicing and the deregulation of this process has a causative role in various human diseases, including cancer. The discovery that alternatively spliced isoforms of several genes are expressed selectively in tumor cells opened the exciting possibility that pharmacological treatment of aberrant splicing could lead to new anti-cancer therapeutic approaches. An alternatively spliced isoform of a scatter factor receptor and proto-oncogene, Ron, accumulates during tumor progression of epithelial tissues and is able to confer an invasive phenotype to the expressing cells. This isoform, called ΔRon, originates from skipping of exon 11, and this specific splicing event is controlled by the expression level of the splicing factor and proto-oncogene SF2/ASF. Over-expression of SF2/ASF, which occurs frequently in various human tumors, induces the production of ΔRon and activates the epithelial to mesenchymal transition (EMT), leading to increased cell motility. In this paper, we have used targeted oligonucleotide enhancers of splicing (TOES) to recruit positive splicing factors to Ron exon 11 and thereby stimulate its inclusion. As an alternative approach, we have used selected indole derivatives that target ASF/SF2 splicing activity. Both treatments correct aberrant ΔRon splicing, restoring the incorporation of Ron exon 11. Notably, indole derivatives are also able to affect the invasive phenotype of the cells. Thus, these treatments may have therapeutic applications for anti-cancer purposes.


Assuntos
Indóis/farmacologia , Metástase Neoplásica/genética , Metástase Neoplásica/terapia , Oligonucleotídeos/farmacologia , Splicing de RNA/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Proto-Oncogene Mas
12.
Diabetes ; 69(4): 647-660, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32005707

RESUMO

Aging-dependent changes in tissue function are associated with the development of metabolic diseases. However, the molecular connections linking aging, obesity, and diabetes remain unclear. Lamin A, lamin C, and progerin, products of the Lmna gene, have antagonistic functions on energy metabolism and life span. Lamin C, albeit promoting obesity, increases life span, suggesting that this isoform is crucial for maintaining healthy conditions under metabolic stresses. Because ß-cell loss during obesity or aging leads to diabetes, we investigated the contribution of lamin C to ß-cell function in physiopathological conditions. We demonstrate that aged lamin C only-expressing mice (Lmna LCS/LCS ) become obese but remain glucose tolerant due to adaptive mechanisms including increased ß-cell mass and insulin secretion. Triggering diabetes in young mice revealed that Lmna LCS/LCS animals normalize their fasting glycemia by both increasing insulin secretion and regenerating ß-cells. Genome-wide analyses combined to functional analyses revealed an increase of mitochondrial biogenesis and global translational rate in Lmna LCS/LCS islets, two major processes involved in insulin secretion. Altogether, our results demonstrate for the first time that the sole expression of lamin C protects from glucose intolerance through a ß-cell-adaptive transcriptional program during metabolic stresses, highlighting Lmna gene processing as a new therapeutic target for diabetes treatment.


Assuntos
Envelhecimento/metabolismo , Diabetes Mellitus/metabolismo , Intolerância à Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Lamina Tipo A/metabolismo , Obesidade/metabolismo , Envelhecimento/genética , Animais , Glicemia/metabolismo , Diabetes Mellitus/genética , Metabolismo Energético/fisiologia , Glucagon/metabolismo , Intolerância à Glucose/genética , Insulina/metabolismo , Lamina Tipo A/genética , Camundongos , Camundongos Transgênicos , Obesidade/genética , Pâncreas/metabolismo
13.
Mol Biol Cell ; 14(12): 4846-56, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12960428

RESUMO

Rho GTPases are key regulators of actin dynamics. We report that the Rho GTPase TCL, which is closely related to Cdc42 and TC10, localizes to the plasma membrane and the early/sorting endosomes in HeLa cells, suggesting a role in the early endocytic pathway. Receptor-dependent internalization of transferrin (Tf) is unaffected by suppression of endogenous TCL by small interfering RNA treatment. However, Tf accumulates in Rab5-positive uncoated endocytic vesicles and fails to reach the early endosome antigen-1-positive early endosomal compartments and the pericentriolar recycling endosomes. Moreover, Tf release upon TCL knockdown is significantly slower. Conversely, in the presence of dominant active TCL, internalized Tf accumulates in early endosome antigen-1-positive early/sorting endosomes and not in perinuclear recycling endosomes. Tf recycles directly from the early/sorting endosomes and it is normally released by the cells. The same phenotype is generated by replacing the C terminus of dominant active Cdc42 and TC10 with that of TCL, indicating that all three proteins share downstream effector proteins. Thus, TCL is essential for clathrin-dependent endocytosed receptors to enter the early/sorting endosomes. Furthermore, the active GTPase favors direct recycling from early/sorting endosomes without accumulating in the perinuclear recycling endosomes.


Assuntos
Membrana Celular/enzimologia , Endocitose/fisiologia , Endossomos/enzimologia , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/efeitos dos fármacos , Células HeLa , Humanos , Imuno-Histoquímica , Ligação Proteica , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , Transferrina/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
14.
Elife ; 52016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27630122

RESUMO

TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53ß promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53ß increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53ß is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53ß depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53ß induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.


Assuntos
Neoplasias da Mama/genética , Neoplasias do Colo/genética , Recidiva Local de Neoplasia/genética , Proteína Supressora de Tumor p53/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias do Colo/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Invasividade Neoplásica/genética , Recidiva Local de Neoplasia/patologia , Isoformas de Proteínas/genética , Proteína Supressora de Tumor p53/biossíntese
15.
Mol Biol Cell ; 24(20): 3164-76, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23966470

RESUMO

Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA-) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.


Assuntos
Processamento Alternativo/genética , Fibronectinas/genética , Invasividade Neoplásica/genética , Proteínas Nucleares/genética , Placentação , Proteínas de Ligação a RNA/genética , Aborto Induzido , Endométrio/crescimento & desenvolvimento , Endométrio/metabolismo , Feminino , Fibronectinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Nucleares/biossíntese , Especificidade de Órgãos/genética , Placenta/metabolismo , Gravidez , Cultura Primária de Células , Proteínas de Ligação a RNA/biossíntese , Fatores de Processamento de Serina-Arginina , Trofoblastos/citologia , Trofoblastos/metabolismo
16.
PLoS One ; 7(11): e48344, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23144867

RESUMO

Rho GTPases are key regulators of tumour cell invasion and therefore constitute attractive targets for the design of anticancer agents. Several strategies have been developed to modulate their increased activities during cancer progression. Interestingly, none of these approaches took into account the existence of the well-known antagonistic relationship between RhoA and Rac1. In this study, we first compared the invasiveness of a collection of colorectal cancer cell lines with their RhoA, Rac1 and Cdc42 activities. A marked decrease of active Cdc42 and Rac1 correlated with the high invasive potential of the cell lines established from metastatic sites of colorectal adenocarcinoma (LoVo, SKCo1, SW620 and CoLo205). Conversely, no correlation between RhoA activity and invasiveness was detected, whereas the activity of its kinase effector ROCK was higher in cancer cell lines with a more invasive phenotype. In addition, invasiveness in these colon cancer cell lines was correlated with a typical round and blebbing morphology. We then tested whether treatment with PDGF to restore Cdc42 and Rac1 activities and/or with Y27632, a chemical inhibitor of ROCK, could decrease the invasiveness of SW620 cells. The association of both treatments substantially decreased the invasive potential of SW620 cells and this effect was accompanied by loss of membrane blebbing, restoration of a more elongated cell morphology and re-establishment of E-cadherin-dependent adherens junctions. This study paves the road to the development of therapeutic strategies in which different Rho GTPase modulators are combined to modulate the cross-talk between Rho GTPases and their specific input in metastatic progression.


Assuntos
Movimento Celular , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Fatores de Despolimerização de Actina/metabolismo , Amidas/farmacologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular , Forma Celular , Humanos , Microscopia de Polarização , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Processamento de Proteína Pós-Traducional , Piridinas/farmacologia , Imagem com Lapso de Tempo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
17.
J Cell Sci ; 117(Pt 26): 6355-64, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15561766

RESUMO

Cell migration is an essential function in various physiological processes, including tissue repair and tumour invasion. Repair of tissue damage requires the recruitment of fibroblasts to sites of tissue injury, which is mediated in part by the cytokine tumour necrosis factor alpha (TNFalpha). As dynamic rearrangements of actin cytoskeleton control cell locomotion, this implicates that TNFalpha is a potent coordinator of cellular actin changes. We have investigated the role of TNFalpha in regulating the cortical actin-containing structures essential for cell locomotion called filopodia. Kinetic analysis of TNFalpha-treated mouse embryonic fibroblasts (MEFs) revealed a dual effect on filopodia formation: a rapid and transient induction mediated by Cdc42 GTPase that is then counteracted by a subsequent sustained inhibition requiring activation of the mitogen-activated protein kinase p38 but not Cdc42 activity. This inhibition also involves the tumour suppressor p53, given that it is activated in response to TNFalpha following the same time course as the decrease of filopodia formation. This functional activation of p53, measured by transcription induction of its target p21WAF1(p21), is also associated with p38 kinase-dependent phosphorylation of p53 at serine 18. Furthermore, TNFalpha did not inhibit filopodia formation in MEFs treated with the transcription inhibitor actinomycin D, in p53-deficient MEFs, or MEFs expressing p53 mutants H273 or H175, which supports a role for the transcriptional activity of p53 in mediating TNFalpha-dependent filopodia inhibition. Our data delineate a novel inhibitory pathway in which TNFalpha prevents filopodia formation and cell migration through the activation of the mitogen-activated protein kinase (MAPK) p38, which in turn activates p53. This shows that TNFalpha on its own initiates antagonistic signals that modulate events linked to cell migration.


Assuntos
Pseudópodes/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Cinética , Camundongos , Fosforilação , Pseudópodes/fisiologia
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