RESUMO
Circulating tumor cells have a strong potential as a quasi-non-invasive tool for setting up a precision medicine strategy for cancer patients. Using a second-generation "filtration-based" technology to isolate CTCs, the Screencell™ technology (Sarcelles, France), we performed a large and simultaneous analysis of all atypical circulating tumor cells (aCTCs) isolated from the blood of metastatic breast cancer (mBC) patients. We correlated their presence with clinicopathological and survival data. We included 91 mBC patients from the PERMED-01 study. The median number of aCTCs was 8.3 per mL of blood. Three subsets of aCTCs, absent from controls, were observed in patients: single (s-aCTCs), circulating tumor micro-emboli (CTM), and giant-aCTCs (g-aCTCs). The presence of g-aCTCs was associated with shorter progression free survival and overall survival. This study highlights the heterogeneity of aCTCs in mBC patients both at the cytomorphological and molecular levels. In addition, it suggests the usefulness of the g-aCTC subset as a prognostic factor and a potential stratification tool to treat late-stage mBC patients and improve their chances of benefiting from early clinical trials.
RESUMO
Cancer treatments are constantly evolving with new approaches to improve patient outcomes. Despite progresses, too many patients remain refractory to treatment due to either the development of resistance to therapeutic drugs and/or metastasis occurrence. Growing evidence suggests that these two barriers are due to transient survival mechanisms that are similar to those observed during stress response. We review the literature and current available open databases to study the potential role of stress response and, most particularly, the involvement of Stress Granules (proteins) in cancer. We propose that Stress Granule proteins may have prognostic value for patients.
RESUMO
Circulating Tumor Cells (CTCs) represent an easy, repeatable and representative access to information regarding solid tumors. However, their detection remains difficult because of their paucity, their short half-life, and the lack of reliable surface biomarkers. Flow cytometry (FC) is a fast, sensitive and affordable technique, ideal for rare cells detection. Adapted to CTCs detection (i.e. extremely rare cells), most FC-based techniques require a time-consuming pre-enrichment step, followed by a 2-hours staining procedure, impeding on the efficiency of CTCs detection. We overcame these caveats and reduced the procedure to less than one hour, with minimal manipulation. First, cells were simultaneously fixed, permeabilized, then stained. Second, using low-speed FC acquisition conditions and two discriminators (cell size and pan-cytokeratin expression), we suppressed the pre-enrichment step. Applied to blood from donors with or without known malignant diseases, this protocol ensures a high recovery of the cells of interest independently of their epithelial-mesenchymal plasticity and can predict which samples are derived from cancer donors. This proof-of-concept study lays the bases of a sensitive tool to detect CTCs from a small amount of blood upstream of in-depth analyses.
Assuntos
Neoplasias da Mama/diagnóstico , Separação Celular/métodos , Neoplasias do Colo/diagnóstico , Citometria de Fluxo/métodos , Células Neoplásicas Circulantes/patologia , Adulto , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Tamanho Celular , Neoplasias do Colo/sangue , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Feminino , Seguimentos , Humanos , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , Prognóstico , Estudo de Prova de Conceito , Estudos Prospectivos , Sensibilidade e Especificidade , Análise de SobrevidaRESUMO
Skeletal dysplasias are a clinically and genetically heterogeneous group of bone and cartilage disorders. A total of 436 skeletal dysplasias are listed in the 2015 revised version of the nosology and classification of genetic skeletal disorders, of which nearly 20% are still genetically and molecularly uncharacterized. We report the clinical and molecular characterization of a lethal skeletal dysplasia of the short-rib group caused by mutation of the mouse Fop gene. Fop encodes a centrosomal and centriolar satellite (CS) protein. We show that Fop mutation perturbs ciliogenesis in vivo and that this leads to the alteration of the Hedgehog signaling pathway. Fop mutation reduces CSs movements and affects pericentriolar material composition, which probably participates to the ciliogenesis defect. This study highlights the role of a centrosome and CSs protein producing phenotypes in mice that recapitulate a short rib-polydactyly syndrome when mutated.
Assuntos
Ciliopatias/genética , Proteínas Proto-Oncogênicas/genética , Síndrome de Costela Curta e Polidactilia/genética , Fatores de Transcrição/genética , Animais , Centríolos/genética , Centrossomo/metabolismo , Centrossomo/patologia , Cílios/genética , Cílios/patologia , Ciliopatias/fisiopatologia , Humanos , Camundongos , Mutação , Síndrome de Costela Curta e Polidactilia/fisiopatologiaRESUMO
A biosensing platform based on an organic transistor circuit for metabolite detection in highly complex biological media is introduced. The sensor circuit provides inherent background subtraction allowing for highly specific, sensitive lactate detection in tumor cell cultures. The proposed sensing platform paves the way toward rapid, label-free, and cost-effective clinically relevant in vitro diagnostic tools.
Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Ácido Láctico/análise , Neoplasias/química , Neoplasias/diagnóstico , Animais , Calibragem , Bovinos , Técnicas de Cultura de Células , Quitosana/química , Impedância Elétrica , Desenho de Equipamento , Compostos Ferrosos/química , Humanos , Leucócitos Mononucleares/química , Metalocenos/química , Oxigenases de Função Mista/química , Poliestirenos , Sensibilidade e Especificidade , Soroalbumina Bovina/química , Tiofenos , Transistores EletrônicosRESUMO
The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs), mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1), recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells.
RESUMO
Cilia and flagella are evolutionary conserved organelles that generate fluid movement and locomotion, and play roles in chemosensation, mechanosensation and intracellular signalling. In complex organisms, cilia are highly diversified, which allows them to perform various functions; however, they retain a 9+0 or 9+2 microtubules structure connected to a basal body. Here, we describe FOR20 (FOP-related protein of 20 kDa), a previously uncharacterized and highly conserved protein that is required for normal formation of a primary cilium. FOR20 is found in PCM1-enriched pericentriolar satellites and centrosomes. FOR20 contains a Lis1-homology domain that promotes self-interaction and is required for its satellite localization. Inhibition of FOR20 expression in RPE1 cells decreases the percentage of ciliated cells and the length of the cilium on ciliated cells. It also modifies satellite distribution, as judged by PCM1 staining, and displaces PCM1 from a detergent-insoluble to a detergent-soluble fraction. The subcellular distribution of satellites is dependent on both microtubule integrity and molecular motor activities. Our results suggest that FOR20 could be involved in regulating the interaction of PCM1 satellites with microtubules and motors. The role of FOR20 in primary cilium formation could therefore be linked to its function in regulating pericentriolar satellites. A role for FOR20 at the basal body itself is also discussed.
Assuntos
Centrossomo/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cílios/metabolismo , Proteínas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Autoantígenos/genética , Autoantígenos/imunologia , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Linhagem Celular Transformada , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Cílios/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Hibridomas , Microtúbulos/metabolismo , Microtúbulos/patologia , Filogenia , Engenharia de Proteínas , Proteínas/genética , RNA Interferente Pequeno/genética , Ratos , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/patologiaRESUMO
FOP is a centrosomal protein originally discovered as a fusion partner of FGFR1 in patients with a rare stem cell myeloproliferative disorder. In DT40 chicken lymphocytes, we show that the normal FOP protein localizes at the centrosome throughout the cell cycle and preferentially accumulates at the distal end of the mother centriole. We used homologous recombination in DT40 cells to generate an inducible null mutant for FOP. Loss of FOP induces apoptosis in the G(1) phase of the cell cycle with accumulation of a 32 kDa P53 tumor suppressor isoform and NOXA and FAS transcripts. However, centrosome integrity and microtubule organization are conserved without FOP and mitotic division and cytokinesis are as efficient as in control cells. Our results suggest that FOP is involved in G(1) to S signaling and thus in proliferation/death fate. Several reports show that centrosome alteration can lead to an arrest in G(1) and, possibly, to senescence in a fraction of cells. The phenotype we observed is more severe in FOP null cells. This could be dependent on the cell context or on the efficiency of a knock out that allows the complete disappearance of the target protein and prevents any de novo synthesis. This is an important observation in regard to the current discussion of what consequence centrosome perturbation could have on a cell and shows that a centrosomal protein can be necessary for cell cycle progression and survival.
Assuntos
Proteínas Aviárias/metabolismo , Ciclo Celular , Centrossomo/metabolismo , Animais , Morte Celular , Linhagem Celular , Sobrevivência Celular , Centríolos/metabolismo , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Galinhas , Fase G1 , Técnicas de Inativação de Genes , Mitose , Mutação/genética , Transporte Proteico , Recombinação Genética/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase lies at the heart of the cell cycle. The APC/C targets mitotic cyclins for destruction in mitosis and G1 phase and is then inactivated at S phase, thereby generating the alternating states of high and low cyclin-Cdk activity required for the alternation of mitosis and DNA replication. Two key questions are how the APC/C is held in check by the spindle-assembly checkpoint to delay cells in mitosis in the presence of improperly attached chromosomes, and how the APC/C is inactivated once cells exit mitosis. The ubiquitin-conjugating protein UbcH10 has been proposed to be crucial in the answers to both questions. However, here we show that the behaviour of UbcH10 is inconsistent with both a crucial role in the spindle checkpoint and in inactivating the APC/C as part of an autonomous oscillator. Instead, we find that the rate-limiting role of UbcH10 is only at the end of G1 phase, just before DNA replication begins.
Assuntos
Fase G1 , Fuso Acromático/enzimologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ciclina A/metabolismo , Células HeLa , Humanos , MitoseRESUMO
JunB, a member of the AP-1 family of dimeric transcription factors, is best known as a cell proliferation inhibitor, a senescence inducer, and a tumor suppressor, although it also has been attributed a cell division-promoting activity. Its effects on the cell cycle have been studied mostly in G1 and S phases, whereas its role in G2 and M phases still is elusive. Using cell synchronization experiments, we show that JunB levels, which are high in S phase, drop during mid- to late G2 phase due to accelerated phosphorylation-dependent degradation by the proteasome. The forced expression of an ectopic JunB protein in late G2 phase indicates that JunB decay is necessary for the subsequent reduction of cyclin A2 levels in prometaphase, the latter event being essential for proper mitosis. Consistently, abnormal JunB expression in late G2 phase entails a variety of mitotic defects. As these aberrations may cause genetic instability, our findings contrast with the acknowledged tumor suppressor activity of JunB and reveal a mechanism by which the deregulation of JunB might contribute to tumorigenesis.
Assuntos
Ciclina A/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Mitose , Fator de Transcrição AP-1/metabolismo , Ciclo Celular , Ciclina A2 , Fase G2 , Vetores Genéticos , Células HeLa , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismoRESUMO
The anaphase promoting complex/cyclosome (APC/C) is crucial to the control of cell division (for a review, see ref. 1). It is a multi-subunit ubiquitin ligase that, at defined points during mitosis, targets specific proteins for proteasomal degradation. The APC/C is itself regulated by the spindle or kinetochore checkpoint, which has an important role in maintaining genomic stability by preventing sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle. The spindle checkpoint regulates the APC/C by inactivating Cdc20, an important co-activator of the APC/C. There is also evidence to indicate that the spindle checkpoint components and Cdc20 are spatially regulated by the mitotic apparatus, in particular they are recruited to improperly attached kinetochores. Here, we show that the APC/C itself co-localizes with components of the spindle checkpoint to improperly attached kinetochores. Indeed, we provide evidence that the spindle checkpoint machinery is required to recruit the APC/C to kinetochores. Our data indicate that the APC/C could be regulated directly by the spindle checkpoint.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Centrômero/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Divisão Celular , Células HeLa , Humanos , Cinetocoros/metabolismo , Ligação Proteica , Transporte Proteico , Fuso Acromático , Transfecção , Complexos Ubiquitina-Proteína Ligase/análiseRESUMO
Prior ubiquitinylation of the unstable c-Fos proto-oncoprotein is thought to be required for recognition and degradation by the proteasome. Contradicting this view, we report that, although c-Fos can form conjugates with ubiquitin in vivo, nonubiquitinylatable c-Fos mutants show regulated degradation identical to that of the wild-type protein in living cells under two classical conditions of study: transient c-fos gene expression during the G(0)/G(1) phase transition upon stimulation by mitogens and constitutive expression during asynchronous growth. Moreover, c-Fos destruction during the G(0)/G(1) phase transition is unusual because it depends on two distinct but cumulative mechanisms. We report here that one mechanism involves a C-terminal destabilizer which does not need an active ubiquitin cycle, whereas the other involves an N-terminal destabilizer dependent on ubiquitinylation of an upstream c-Fos breakdown effector. In addition to providing new insights into the mechanisms of c-Fos protein destruction, an important consequence of our work is that ubiquitinylation-dependent proteasomal degradation claimed for a number of proteins should be reassessed on a new experimental basis.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ubiquitina/metabolismo , Divisão Celular , Fase G1 , Células HeLa , Humanos , Immunoblotting , Modelos Biológicos , Mutagênese Sítio-Dirigida , Fenótipo , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Estrutura Terciária de Proteína , Fase de Repouso do Ciclo Celular , Temperatura , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
c-fos gene is expressed constitutively in a number of tissues as well as in certain tumor cells and is inducible, in general rapidly and transiently, in virtually all other cell types by a variety of stimuli. Its protein product, c-Fos, is a short-lived transcription factor that heterodimerizes with various protein partners within the AP-1 transcription complex via leucine zipper/leucine zipper interactions for binding to specific DNA sequences. It is mostly, if not exclusively, degraded by the proteasome. To localize the determinant(s) responsible for its instability, we have conducted a genetic analysis in which the half-lives of c-Fos mutants and chimeras made with the stable EGFP reporter protein were compared under two experimental conditions taken as example of continous and inducible expression. Those were constitutive expression in asynchronously growing Balb/C 3T3 mouse embryo fibroblasts and transient induction in the same cells undergoing the G0/G1 phase transition upon stimulation by serum. Our work shows that c-Fos is degraded faster in synchronous- than in asynchronous cells. This difference in turnover is primarily accounted for by several mechanisms. First, in asynchronous cells, a unique C-terminal destabilizer is active whereas, in serum-stimulated cells two destabilizers located at both extremities of the protein are functional. Second, heterodimerization and/or binding to DNA accelerates protein degradation only during the G0/G1 phase transition. Adding another level of complexity to turnover control, phosphorylation at serines 362 and 374, which are c-Fos phosphorylation sites largely modified during the G0/G1 phase transition, stabilizes c-Fos much more efficiently in asynchronous than in serum-stimulated cells. In both cases, the reduced degradation rate is due to inhibition of the activity of the C-terminal destabilizer. However, in serum-stimulated cells, this effect is partially masked by the activation of the N-terminal destabilizer and basic domain/leucine zipper-dependent mechanisms. Taken together, our data show that multiple degradation mechanisms, differing according to the conditions of expression, may operate on c-Fos to ensure a proper level and/or timing of expression. Moreover, they also indicate that the half-life of c-Fos during the G0/G1 phase transition is determined by a delicate balance between opposing stabilizing and destabilizing mechanisms operating at the same time.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Divisão Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Fator de Crescimento Epidérmico/genética , Fibroblastos , Fase G1/fisiologia , Regulação da Expressão Gênica , Genes Reporter , Meia-Vida , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Serina/metabolismoRESUMO
c-Fos protooncoprotein is a short-lived transcription factor with oncogenic potential. It is massively degraded by the proteasome in vivo under various experimental conditions. Those include consititutive expression in exponentially growing cells and transient induction in cells undergoing the G0/G1 phase transition upon stimulation by serum. Though there is evidence that c-Fos can be ubiquitinylated in vitro, the unambigous demonstration that prior ubiquitinylation is necessary for degradation by the proteasome in vivo is still lacking. c-Jun, one of the main dimerization partners of c-Fos within the AP-1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome dependent. However, several lines of evidence indicate that the mechanisms by which it addresses the proteasome are different from those operating on c-Fos. Moreover, genetic analysis has indicated that c-Fos is addressed to the proteasome via pathways that differ depending on the conditions of expression. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses in mutated forms, which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of one of the main c-Fos destabilizers but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. However, although viral Fos proteins have acquired full resistance to proteasomal degradation, stabilization is limited because the mutations they have accumulated, during or after c-fos gene transduction, confer sensitivity to an unidentified proteolytic system(s). This observation is consistent with the idea that fos-expressing viruses have evolved expression machineries to ensure controlled protein levels in order to maintain an optimal balance between prooncogenic and proapoptotic activities of v-Fos proteins.
Assuntos
Proteínas Oncogênicas v-fos/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular/fisiologia , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica , Humanos , Complexos Multienzimáticos/metabolismo , Fragmentos de Peptídeos/química , Complexo de Endopeptidases do Proteassoma , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismoRESUMO
c-Fos proto-oncoprotein is highly unstable, which is crucial for rapid gene expression shut-off and control of its intrinsic oncogenic potential. It is massively degraded by the proteasome in vivo in various situations. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for recognition and subsequent hydrolysis by the proteasome in vivo is still lacking. Moreover, genetic analysis have also indicated that c-Fos can be addressed to the proteasome via different mechanisms depending on the conditions studied. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of a C-terminal c-Fos destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to (an) unidentified proteolytic system(s). This observation is consistent with the idea that Fos-expressing viruses have evolved gene expression controls that avoid high protein accumulation-linked apoptosis.