RESUMO
The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.
Assuntos
Estômago/citologia , Proteínas de Sinalização YAP/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Ectoderma/citologia , Ectoderma/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Proteína Nodal/antagonistas & inibidores , Proteína Nodal/genética , Proteína Nodal/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Estômago/metabolismo , Proteínas de Sinalização YAP/deficiência , Proteínas de Sinalização YAP/genéticaRESUMO
Too much of a good thing can become a bad thing. An example is FBXW7, a well-known tumor suppressor that may also contribute to tumorigenesis. Here, we reflect on the results of three laboratories describing the role of FBXW7 in the degradation of p53 and the possible implications of this finding in tumor cell development. We also speculate about the function of FBXW7 as a key player in the cell fate after DNA damage and how this could be exploited in the treatment of cancer disease.
RESUMO
Cyclin E, a key cell cycle regulatory protein, has been linked to oncogenesis when dysregulated. We have previously shown that overexpression of cyclin E causes replication stress, leading to failure to complete replication at specific chromosomal loci during S phase of the cell cycle. This in turn promotes chromosomal damage during anaphase. Here we show that non-transformed human mammary epithelial cell clones that survive such aberrant mitoses have a specific and reproducible pattern of chromosomal Copy Number Alterations (CNAs) that we have characterized and termed the cyclin E CNA signature. Using a number of computational approaches, we show that this signature resembles one specific CNA pattern enriched in differentiated epithelial-like tumors of the breast and ovary. Analysis of the CNA profile of these clones provides a potential mechanism for cyclin E-mediated oncogenesis.
RESUMO
Eukaryotic cells have developed sophisticated mechanisms to ensure the integrity of the genome and prevent the transmission of altered genetic information to daughter cells. If this control system fails, accumulation of mutations would increase risk of diseases such as cancer. Ubiquitylation, an essential process for protein degradation and signal transduction, is critical for ensuring genome integrity as well as almost all cellular functions. Here, we investigated the role of the SKP1-Cullin-1-F-box protein (SCF)-[F-box and tryptophan-aspartic acid (WD) repeat domain containing 7 (FBXW7)] ubiquitin ligase in cell proliferation by searching for targets implicated in this process. We identified a hitherto-unknown FBXW7-interacting protein, p53, which is phosphorylated by glycogen synthase kinase 3 at serine 33 and then ubiquitylated by SCF(FBXW7) and degraded. This ubiquitylation is carried out in normally growing cells but primarily after DNA damage. Specifically, we found that SCF(FBXW7)-specific targeting of p53 is crucial for the recovery of cell proliferation after UV-induced DNA damage. Furthermore, we observed that amplification of FBXW7 in wild-type p53 tumors reduced the survival of patients with breast cancer. These results provide a rationale for using SCF(FBXW7) inhibitors in the treatment of this subset of tumors.-Galindo-Moreno, M., Giráldez, S., Limón-Mortés, M. C., Belmonte-Fernández, A., Reed, S. I., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. SCF(FBXW7)-mediated degradation of p53 promotes cell recovery after UV-induced DNA damage.
Assuntos
Dano ao DNA/genética , Proteína 7 com Repetições F-Box-WD/genética , Proteína Supressora de Tumor p53/genética , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Chlorocebus aethiops , Proteínas F-Box/genética , Células HCT116 , Células HEK293 , Humanos , Mutação/genética , Fosforilação/genética , Domínios Proteicos/genética , Proteólise , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
Cyclin-dependent kinase 1 (CDK1) is the central mammalian regulator of cell proliferation and a promising therapeutic target for breast cancer. In fact, CDK1 inhibition downregulates survival and induces apoptosis. Due to its essential role, CDK1 expression and activity are strictly controlled at various levels. We previously described that CDK1 stability is also regulated and that SCF(ßTrCP) ubiquitinates CDK1, which is degraded via the lysosomal pathway. In addition, in breast tumors from patients, we found a negative correlation between CDK1 accumulation and ßTrCP levels, and a positive correlation with the degree of tumor malignancy. This prompted us to study the molecular mechanism involved in CDK1 clearance. In this report, we determine that both chemotherapeutic agents and proteolytic stress induce CDK1 degradation in human breast cancer MCF7 cells through p62/HDAC6-mediated selective autophagy. On the one hand, CDK1 binds to p62/SQSTM1-LC3 and, on the other hand, it interacts with HDAC6. Both complexes are dependent on the presence of an intact ßTrCP-binding motif on CDK1. Furthermore, we also show that CDK1 is recruited to aggresomes in response to proteasome inhibition for an extended period. We propose CDK1 clearance as a potential predictive biomarker of antitumor treatment efficacy.
Assuntos
Autofagia/genética , Proteína Quinase CDC2/genética , Regulação Neoplásica da Expressão Gênica , Desacetilase 6 de Histona/genética , Proteínas Ligases SKP Culina F-Box/genética , Proteína Sequestossoma-1/genética , Proteína Quinase CDC2/metabolismo , Células HEK293 , Células HeLa , Desacetilase 6 de Histona/metabolismo , Humanos , Células MCF-7 , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , UbiquitinaçãoRESUMO
Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCFßTrCP/proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G1/S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCFßTrCP/proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G1-to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G1/S phase progression is regulated by PLK1 degradation through the CDK1/ßTrCP axis.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Plasmídeos , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas Contendo Repetições de beta-Transducina/genética , Quinase 1 Polo-LikeRESUMO
FBXW7 is a component of SCF (complex of SKP1, CUL1 and F-box-protein)-type ubiquitin ligases that targets several oncoproteins for ubiquitination and degradation by the proteasome. FBXW7 regulates cellular apoptosis by targeting MCL1 for ubiquitination. Recently, we identified PLK1 as a new substrate of FBXW7 modulating the intra-S-phase DNA-damage checkpoint. Taxanes are frequently used in breast cancer treatments, but the acquisition of resistance makes these treatments ineffective. We investigated the role of FBXW7 and their substrates MCL1 and PLK1 in regulating the apoptotic response to paclitaxel treatment in breast cancer cells and their expression in breast cancer tissues. Paclitaxel-sensitive MDA-MB-468 and a paclitaxel-resistant MDA-MB-468R subclone were used to study the role of FBXW7 and substrates in paclitaxel-induced apoptosis. Forced expression of FBXW7 or downregulation of MCL1 or PLK1 restored sensitivity to paclitaxel in MDA-MB-468R cells. By contrary, FBXW7-silenced MDA-MB-468 cells became resistant to paclitaxel. The expression of FBXW7 and substrates were studied in 296 invasive carcinomas by immunohistochemistry and disease-free survival was analyzed in a subset of patients treated with paclitaxel. In breast cancer tissues, loss of FBXW7 correlated with adverse prognosis markers and loss of FBXW7 and MCL1 or PLK1 accumulation were associated with diminished disease-free survival in paclitaxel-treated patients. We conclude that FBXW7 regulates the response to paclitaxel by targeting MCL1 and PLK1 in breast cancer cells and thus targeting these substrates may be a valuable adjunct for paclitaxel treatment. Also, FBXW7, MCL1 and PLK1 may be relevant predictive markers of tumor progression and response to paclitaxel treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Proteína 7 com Repetições F-Box-WD/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Paclitaxel/administração & dosagem , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Quinase 1 Polo-LikeRESUMO
Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3ß (GSK-3ß) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3ß behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3ß and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular , Autofagia , Proliferação de Células , Glicogênio Sintase Quinase 3 beta , Humanos , Células MCF-7 , Transdução de SinaisRESUMO
In mammals, cell cycle progression is controlled by cyclin-dependent kinases, among which CDK1 plays important roles in the regulation of the G2/M transition, G1 progression and G1/S transition. CDK1 is highly regulated by its association to cyclins, phosphorylation and dephosphorylation, changes in subcellular localization, and by direct binding of CDK inhibitor proteins. CDK1 steady-state protein levels are held constant throughout the cell cycle by a coordinated regulation of protein synthesis and degradation. We show that CDK1 is ubiquitinated by the E3 ubiquitin ligase SCFßTrCP and degraded by the lysosome. Furthermore, we found that DNA damage not only triggers the stabilization of inhibitory phosphorylation sites on CDK1 and repression of CDK1 gene expression, but also regulates ßTrCP-induced CDK1 degradation in a cell type-dependent manner. Specifically, treatment with the chemotherapeutic agent doxorubicin in certain cell lines provokes CDK1 degradation and induces apoptosis, whereas in others it inhibits destruction of the protein. These observations raise the possibility that different tumor types, depending on their pathogenic spectrum mutations, may display different sensitivity to ßTrCP-induced CDK1 degradation after DNA damage. Finally, we found that CDK1 accumulation in patients' tumors shows a negative correlation with ßTrCP and a positive correlation with the degree of tumor malignancy.
Assuntos
Proteína Quinase CDC2/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Animais , Western Blotting , Linhagem Celular , Humanos , Imunoprecipitação , Lisossomos/metabolismo , Invasividade Neoplásica/patologia , RNA Interferente Pequeno , Espectrometria de Massas em Tandem , Análise Serial de Tecidos , TransfecçãoRESUMO
The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Cromatografia Líquida , Dano ao DNA , Proteína 7 com Repetições F-Box-WD , Células HeLa , Humanos , Camundongos , Fase S , Espectrometria de Massas em Tandem , Transfecção , Quinase 1 Polo-LikeRESUMO
Pituitary tumour transforming gene (pttg1) encodes Securin, a protein involved in the inhibition of sister chromatid separation binding to Separase until the onset of anaphase. Separase is a cysteine-protease that degrades cohesin to segregate the sister chromatids to opposite poles of the cell. The amount of Securin is strongly regulated because it should allow Separase activation when it is degraded by the anaphase promoting complex/cyclosome, should arrest the cell cycle after DNA damage, when it is degraded through SKP1-CUL1-ßTrCP ubiquitin ligase, and its overexpression induces tumour formation and correlates with metastasis in multiple tumours. Securin is a phosphoprotein that contains 32 potentially phosphorylatable residues. We mutated and analysed most of them, and found a single mutant, hSecT60A, that showed enhanced oncogenic properties. Our fluorescence activated cell sorting analysis, fluorescence in situ hybridisation assays, tumour cell migration and invasion experiments and gene expression by microarrays analysis clearly involved hSecT60A in chromosomal instability and cell invasion. These results show, for the first time, that a single mutation in pttg1 is sufficient to trigger the oncogenic properties of Securin. The finding of this point mutation in patients might be used as an effective strategy for early detection of cancer.
Assuntos
Transformação Celular Neoplásica/genética , Instabilidade Cromossômica , Proteínas de Neoplasias/genética , Neoplasias/genética , Mutação Puntual , Animais , Células COS , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Chlorocebus aethiops , Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Análise em Microsséries , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Securina , Treonina/genética , TransfecçãoRESUMO
PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3ß (GSK3ß) phosphorylates securin to promote its proteolysis via SCF(ßTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3ß inactivation was observed in breast cancer tissues, indicating that GSK3ß inactivation may account for securin accumulation in breast cancers.