RESUMEN
The AKT-mTOR pathway is a central regulator of cell growth and metabolism. Upon sustained mTOR activity, AKT activity is attenuated by a feedback loop that restrains upstream signaling. However, how cells control the signals that limit AKT activity is not fully understood. Here, we show that MASTL/Greatwall, a cell cycle kinase that supports mitosis by phosphorylating the PP2A/B55 inhibitors ENSA/ARPP19, inhibits PI3K-AKT activity by sustaining mTORC1- and S6K1-dependent phosphorylation of IRS1 and GRB10. Genetic depletion of MASTL results in an inefficient feedback loop and AKT hyperactivity. These defects are rescued by the expression of phosphomimetic ENSA/ARPP19 or inhibition of PP2A/B55 phosphatases. MASTL is directly phosphorylated by mTORC1, thereby limiting the PP2A/B55-dependent dephosphorylation of IRS1 and GRB10 downstream of mTORC1. Downregulation of MASTL results in increased glucose uptake in vitro and increased glucose tolerance in adult mice, suggesting the relevance of the MASTL-PP2A/B55 kinase-phosphatase module in controlling AKT and maintaining metabolic homeostasis.
Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina , Proteína Fosfatasa 2 , Proteínas Serina-Treonina Quinasas , Animales , Ratones , Ciclo Celular/genética , Glucosa/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mitosis , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human-mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.
Asunto(s)
Diferenciación Celular , Metilación de ADN , Epigénesis Genética , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/metabolismo , Animales , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , MicroARNs/genética , ADN Metiltransferasa 3BRESUMEN
Activation of WNT/ß-catenin signaling has been associated with a non-T-cell-inflamed tumor microenvironment (TME) in several cancers. The aim of this work was to investigate the relationship between ß-catenin signaling and TME inflammation in head and neck squamous cell carcinomas (HNSCCs). Membrane and nuclear ß-catenin expression, PD-L1 expression, and CD8+ tumor-infiltrating lymphocyte (TIL) density were jointly evaluated by immunohistochemistry in a series of 372 HPV-negative HNSCCs. Membrane ß-catenin levels decreased in carcinomas compared to the normal epithelium. Positive nuclear ß-catenin was detected in 50 tumors (14.3%) and was significantly associated with a low CD8+ TIL density (168 cells/mm2 versus 293 cells/mm2 in nuclear-ß-catenin-negative cases; p = 0.01) and a tendency for a lower expression of PD-L1, resulting in association with a noninflamed TME (i.e., type II, immunological ignorance). Multivariate Cox analysis further demonstrated that low infiltration by CD8+ TILs (HR = 1.6, 95% CI = 1.19-2.14, p = 0.002) and nuclear ß-catenin expression (HR = 1.47, 95% CI = 1.01-2.16, p = 0.04) were both independently associated with a poorer disease-specific survival. In conclusion, tumor-intrinsic nuclear ß-catenin activation is associated with a non-inflamed TME phenotype and a poorer prognosis, thereby suggesting a possible implication as an immune exclusion mechanism for a subset of HNSCC patients.
Asunto(s)
Antígeno B7-H1 , Neoplasias de Cabeza y Cuello , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Linfocitos Infiltrantes de Tumor , Fenotipo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Microambiente Tumoral , beta Catenina/metabolismoRESUMEN
The cellular response to DNA damage is essential for maintaining the integrity of the genome. Recent evidence has identified E2F7 as a key player in DNA damage-dependent transcriptional regulation of cell-cycle genes. However, the contribution of E2F7 to cellular responses upon genotoxic damage is still poorly defined. Here we show that E2F7 represses the expression of genes involved in the maintenance of genomic stability, both throughout the cell cycle and upon induction of DNA lesions that interfere with replication fork progression. Knockdown of E2F7 leads to a reduction in 53BP1 and FANCD2 foci and to fewer chromosomal aberrations following treatment with agents that cause interstrand crosslink (ICL) lesions but not upon ionizing radiation. Accordingly, E2F7-depleted cells exhibit enhanced cell-cycle re-entry and clonogenic survival after exposure to ICL-inducing agents. We further report that expression and functional activity of E2F7 are p53-independent in this context. Using a cell-based assay, we show that E2F7 restricts homologous recombination through the transcriptional repression of RAD51. Finally, we present evidence that downregulation of E2F7 confers an increased resistance to chemotherapy in recombination-deficient cells. Taken together, our results reveal an E2F7-dependent transcriptional program that contributes to the regulation of DNA repair and genomic integrity.
Asunto(s)
Reparación del ADN , Factor de Transcripción E2F7/fisiología , Regulación de la Expresión Génica , Inestabilidad Genómica , Ciclo Celular/genética , Línea Celular , Rotura Cromosómica , Daño del ADN , Factor de Transcripción E2F7/metabolismo , Humanos , Regiones Promotoras Genéticas , Reparación del ADN por Recombinación , Transcripción Genética , Transcriptoma , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Chromosome segregation requires the ordered separation of the newly replicated chromosomes between the two daughter cells. In most cells, this requires nuclear envelope (NE) disassembly during mitotic entry and its reformation at mitotic exit. Nuclear envelope breakdown (NEB) results in the mixture of two cellular compartments. This process is controlled through phosphorylation of multiple targets by cyclin-dependent kinase 1 (Cdk1)-cyclin B complexes as well as other mitotic enzymes. Experimental evidence also suggests that nucleo-cytoplasmic transport of critical cell cycle regulators such as Cdk1-cyclin B complexes or Greatwall, a kinase responsible for the inactivation of PP2A phosphatases, plays a major role in maintaining the boost of mitotic phosphorylation thus preventing the potential mitotic collapse derived from NEB. These data suggest the relevance of nucleo-cytoplasmic transport not only to communicate cytoplasmic and nuclear compartments during interphase, but also to prepare cells for the mixture of these two compartments during mitosis.
Asunto(s)
Mitosis , Membrana Nuclear/metabolismo , Animales , Segregación Cromosómica , Humanos , Proteínas Asociadas a Microtúbulos/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/fisiología , Transporte de ProteínasRESUMEN
Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Membrana Nuclear/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Mamíferos/embriología , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Ratones Noqueados , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Cell cycle progression is regulated by the orderly balance between kinase and phosphatase activities. PP2A phosphatase holoenzymes containing the B55 family of regulatory B subunits function as major CDK1-counteracting phosphatases during mitotic exit in mammals. However, the identification of the specific mitotic roles of these PP2A-B55 complexes has been hindered by the existence of multiple B55 isoforms. Here, through the generation of loss-of-function genetic mouse models for the two ubiquitous B55 isoforms (B55α and B55δ), we report that PP2A-B55α and PP2A-B55δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55 activity, mitotic cells display increased chromosome individualization in the presence of enhanced phosphorylation and perichromosomal loading of Ki-67. These data provide experimental evidence for a regulatory mechanism by which the balance between kinase and PP2A-B55 phosphatase activity controls the Ki-67-mediated spatial organization of the mass of chromosomes during mitosis.
Asunto(s)
Antígeno Ki-67 , Mitosis , Proteína Fosfatasa 2 , Animales , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genética , Ratones , Antígeno Ki-67/metabolismo , Fosforilación , Cromosomas de los Mamíferos/metabolismo , Cromosomas de los Mamíferos/genética , Cromosomas/metabolismoRESUMEN
Metastatic prostate cancer (PCa) inevitably acquires resistance to standard therapy preceding lethality. Here, we unveil a chromosomal instability (CIN) tolerance mechanism as a therapeutic vulnerability of therapy-refractory lethal PCa. Through genomic and transcriptomic analysis of patient datasets, we find that castration and chemotherapy-resistant tumors display the highest CIN and mitotic kinase levels. Functional genomics screening coupled with quantitative phosphoproteomics identify MASTL kinase as a survival vulnerability specific of chemotherapy-resistant PCa cells. Mechanistically, MASTL upregulation is driven by transcriptional rewiring mechanisms involving the non-canonical transcription factors androgen receptor splice variant 7 and E2F7 in a circuitry that restrains deleterious CIN and prevents cell death selectively in metastatic therapy-resistant PCa cells. Notably, MASTL pharmacological inhibition re-sensitizes tumors to standard therapy and improves survival of pre-clinical models. These results uncover a targetable mechanism promoting high CIN adaptation and survival of lethal PCa.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Inestabilidad Cromosómica , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/uso terapéutico , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The forkhead transcription factor FoxM1 controls expression of a large number of genes that are specifically expressed during the G(2) phase of the cell cycle. Throughout most of the cell cycle, FoxM1 activity is restrained by an autoinhibitory mechanism, involving a repressor domain present in the N-terminal part of the protein. Activation of FoxM1 in G(2) is achieved by Cyclin A/Cyclin-dependent kinase (Cdk)-mediated phosphorylation, which alleviates autoinhibition by the N-terminal repressor domain. Here, we show that FoxM1 interacts with B55α, a regulatory subunit of protein phosphatase 2A (PP2A). B55α binds the catalytic subunit of PP2A, and this promotes dephosphorylation and inactivation of FoxM1. Indeed, we find that overexpression of B55α results in decreased FoxM1 activity. Inversely, depletion of B55α results in premature activation of FoxM1. The activation of FoxM1 that is observed upon depletion of B55α is fully dependent on Cyclin A/Cdk-mediated phosphorylation of FoxM1. Taken together, these data demonstrate that B55α acts to antagonize Cyclin A/Cdk-dependent activation of FoxM1, to ensure that FoxM1 activity is restricted to the G(2) phase of the cell cycle.
Asunto(s)
Ciclina A/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteína Fosfatasa 2/metabolismo , Secuencia de Aminoácidos , Línea Celular , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Humanos , Datos de Secuencia Molecular , Péptidos/química , Fosforilación , Unión Proteica , Transcripción GenéticaRESUMEN
Activation of the DNA-damage checkpoint culminates in the inhibition of cyclin-dependent kinase (Cdk) complexes to prevent cell-cycle progression. We have shown recently that Cdk activity is required for activation of the Forkhead transcription factor FoxM1, an important regulator of gene expression in the G2 phase of the cell cycle. Here, we show that FoxM1 is transcriptionally active during a DNA-damage-induced G2 arrest and is essential for checkpoint recovery. Paradoxically, Cdk activity, although reduced after checkpoint activation, is required to maintain FoxM1-dependent transcription during the arrest and for expression of pro-mitotic targets such as cyclin A, cyclin B and Plk1. Indeed, we find that cells need to retain sufficient levels of Cdk activity during the DNA-damage response to maintain cellular competence to recover from a DNA-damaging insult.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Daño del ADN , Factores de Transcripción Forkhead/genética , Fase G2 , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/metabolismo , Humanos , Transcripción GenéticaRESUMEN
Inhibiting the cell-cycle kinases CDK4 and CDK6 results in significant therapeutic effect in patients with advanced hormone-positive breast cancer. The efficacy of this strategy is, however, limited by innate or acquired resistance mechanisms and its application to other tumor types is still uncertain. Here, through an integrative analysis of sensitivity and resistance mechanisms, we discuss the use of CDK4/6 inhibitors in combination with available targeted therapies, immunotherapy, or classical chemotherapy with the aim of improving future therapeutic uses of CDK4/6 inhibition in a variety of cancers.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Humanos , Neoplasias/enzimología , Neoplasias/patologíaRESUMEN
Cancer stem cells (CSCs) play major roles in tumor initiation, progression, and resistance to cancer therapy. Several CSC markers have been studied in head and neck squamous cell carcinomas (HNSCC), including the pluripotency factors NANOG, SOX2, and OCT4; however, their clinical significance is still unclear. NANOG, SOX2, and OCT4 expression was evaluated by immunochemistry in 348 surgically-treated HNSCC, and correlated with clinicopathological parameters and patient outcomes. mRNA expression was further analyzed in 530 The Cancer Genome Atlas (TCGA) HNSCC. NANOG protein expression was detected in 250 (72%) cases, more frequently in patients with lymph node metastasis (p = 0.003), and was an independent predictor of better survival in multivariate analysis. While OCT4 expression was undetectable, SOX2 expression was observed in 105 (30%) cases, and strongly correlated with NANOG expression. Combined expression of both proteins showed the highest survival rates, and double-negative cases the worst survival. Strikingly, the impact of NANOG and SOX2 on outcome varied depending on tumor site and lymph node infiltration, specifically showing prognostic significance in pharyngeal tumors. Correlation between NANOG and SOX2 at mRNA and protein was specifically observed in node positive (N+) patients, and consistently correlated with better survival rates. According to our findings, NANOG protein expression is frequent in HNSCC, thereby emerging as an independent predictor of better prognosis in pharyngeal tumors. Moreover, this study uncovers a differential impact of NANOG and SOX2 expression on HNSCC prognosis, depending on tumor site and lymph node infiltration, which could facilitate high-risk patient stratification.
RESUMEN
OBJECTIVES: The function of NOTCH signaling (oncogenic or oncosuppressive) remains controversial in head and neck squamous cell carcinomas (HNSCC). The purpose of this work is to investigate the role of NOTCH pathway in HNSCC prognosis. METHODS: Immunohistochemical NOTCH1 and HES1 expression was jointly evaluated and correlated with other NOTCH1 targets, p21 (WAF1/Cip1) and Cyclin D1, using an unbiased cohort of 372 surgically treated HPV-negative HNSCC patients. RESULTS: Membranous NOTCH1 expression was detected in 197 (61%) out of 324 evaluable tumor samples, and nuclear NOTCH1 expression in 91 samples (28%). Nuclear HES1 expression was found in 224 (67%) cases. Membranous and nuclear NOTCH1 expression were consistently and significantly correlated with nuclear HES1 (P < 0.001) and p21 (Pâ¯=â¯0.03) expression, but not with Cyclin D1. NOTCH1 expression was significantly associated to early stages (I-II), non-recurrent disease, and better disease-specific (DSS) and overall survival (OS) rates (P < 0.001). Moreover, triple-positive cases (NOTCH1+/HES1+/p21+) exhibited significantly improved DSS (P < 0.001) and OS (P = 0.004), thus reinforcing the association of NOTCH pathway activation with a better prognosis in HNSCC. Multivariate analysis further revealed membranous NOTCH1 expression as a robust independent predictor of better DSS (HR = 0.554; 95% IC 0.412-0.745; P < 0.001) and better OS (HR = 0.640; 95% CI 0.491-0.835; P = 0.001). CONCLUSION: These findings show the association of NOTCH pathway activation with a better prognosis in HNSCC patients, also revealing membranous NOTCH1 expression as a robust independent predictor of improved survival. Accordingly, these results suggest a tumor suppressive rather than an oncogenic role for NOTCH pathway in HNSCC.
Asunto(s)
Receptores Notch/metabolismo , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Línea Celular Tumoral , Biología Computacional/métodos , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Modelos de Riesgos Proporcionales , Receptores Notch/genética , Recurrencia , Carcinoma de Células Escamosas de Cabeza y Cuello/diagnóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/etiología , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismoRESUMEN
Inhibition of the cell-cycle kinases CDK4 and CDK6 is now part of the standard treatment in advanced breast cancer. CDK4/6 inhibitors, however, are not expected to cooperate with DNA-damaging or antimitotic chemotherapies as the former prevent cell-cycle entry, thus interfering with S-phase- or mitosis-targeting agents. Here, we report that sequential administration of CDK4/6 inhibitors after taxanes cooperates to prevent cellular proliferation in pancreatic ductal adenocarcinoma (PDAC) cells, patient-derived xenografts, and genetically engineered mice with Kras G12V and Cdkn2a-null mutations frequently observed in PDAC. This effect correlates with the repressive activity of CDK4/6 inhibitors on homologous recombination proteins required for the recovery from chromosomal damage. CDK4/6 inhibitors also prevent recovery from multiple DNA-damaging agents, suggesting broad applicability for their sequential administration after available chemotherapeutic agents.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Albúminas/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Reparación del ADN/efectos de los fármacos , Esquema de Medicación , Recombinación Homóloga/efectos de los fármacos , Humanos , Ratones Desnudos , Ratones Transgénicos , Mutación , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Paclitaxel/administración & dosificación , Neoplasias Pancreáticas/patología , Piperazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridinas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell-cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyperstabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyperphosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.
Asunto(s)
Citoesqueleto de Actina , Plaquetas/enzimología , Rotura Cromosómica , Trastornos de los Cromosomas , Proteínas Asociadas a Microtúbulos , Mutación Missense , Proteínas Serina-Treonina Quinasas , Transducción de Señal/genética , Trombocitopenia/congénito , Citoesqueleto de Actina/enzimología , Citoesqueleto de Actina/genética , Sustitución de Aminoácidos , Animales , Plaquetas/patología , Trastornos de los Cromosomas/enzimología , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/patología , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Trombocitopenia/enzimología , Trombocitopenia/genética , Trombocitopenia/patologíaRESUMEN
PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.
Asunto(s)
Neoplasias de la Mama/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas de Neoplasias/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteína Fosfatasa 2/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Distinct protein phosphorylation levels in interphase and M phase require tight regulation of Cdk1 activity [1, 2]. A bistable switch, based on positive feedback in the Cdk1 activation loop, has been proposed to generate different thresholds for transitions between these cell-cycle states [3-5]. Recently, the activity of the major Cdk1-counteracting phosphatase, PP2A:B55, has also been found to be bistable due to Greatwall kinase-dependent regulation [6]. However, the interplay of the regulation of Cdk1 and PP2A:B55 in vivo remains unexplored. Here, we combine quantitative cell biology assays with mathematical modeling to explore the interplay of mitotic kinase activation and phosphatase inactivation in human cells. By measuring mitotic entry and exit thresholds using ATP-analog-sensitive Cdk1 mutants, we find evidence that the mitotic switch displays hysteresis and bistability, responding differentially to Cdk1 inhibition in the mitotic and interphase states. Cdk1 activation by Wee1/Cdc25 feedback loops and PP2A:B55 inactivation by Greatwall independently contributes to this hysteretic switch system. However, elimination of both Cdk1 and PP2A:B55 inactivation fully abrogates bistability, suggesting that hysteresis is an emergent property of mutual inhibition between the Cdk1 and PP2A:B55 feedback loops. Our model of the two interlinked feedback systems predicts an intermediate but hidden steady state between interphase and M phase. This could be verified experimentally by Cdk1 inhibition during mitotic entry, supporting the predictive value of our model. Furthermore, we demonstrate that dual inhibition of Wee1 and Gwl kinases causes loss of cell-cycle memory and synthetic lethality, which could be further exploited therapeutically.
Asunto(s)
Ciclo Celular , Mitosis , Ciclo Celular/genética , Células HeLa , Humanos , Interfase/genética , Mitosis/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , FosforilaciónRESUMEN
The alterations in carbohydrate metabolism that fuel tumor growth have been extensively studied. However, other metabolic pathways involved in malignant progression, demand further understanding. Here we describe a metabolic acyl-CoA synthetase/stearoyl-CoA desaturase ACSL/SCD network causing an epithelial-mesenchymal transition (EMT) program that promotes migration and invasion of colon cancer cells. The mesenchymal phenotype produced upon overexpression of these enzymes is reverted through reactivation of AMPK signaling. Furthermore, this network expression correlates with poorer clinical outcome of stage-II colon cancer patients. Finally, combined treatment with chemical inhibitors of ACSL/SCD selectively decreases cancer cell viability without reducing normal cells viability. Thus, ACSL/SCD network stimulates colon cancer progression through conferring increased energetic capacity and invasive and migratory properties to cancer cells, and might represent a new therapeutic opportunity for colon cancer treatment.