RESUMO
AIMS/HYPOTHESIS: The conserved hypoxia inducible factor 1 α (HIF1α) injury-response pro-survival pathway has recently been implicated in early beta cell dysfunction but slow beta cell loss in type 2 diabetes. We hypothesised that the unexplained prolonged prediabetes phase in type 1 diabetes may also be, in part, due to activation of the HIF1α signalling pathway. METHODS: RNA sequencing (RNA-Seq) data from human islets with type 1 diabetes or after cytokine exposure in vitro was evaluated for activation of HIF1α targets. This was corroborated by immunostaining human pancreases from individuals with type 1 diabetes for 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), the key effector of HIF1α-mediated metabolic remodelling, and by western blotting of islets and INS-1 832/13 cells exposed to cytokines implicated in type 1 diabetes. RESULTS: HIF1α signalling is activated (p = 4.5 × 10-9) in islets from individuals with type 1 diabetes, and in human islets exposed in vitro to cytokines implicated in type 1 diabetes (p = 1.1 × 10-14). Expression of PFKFB3 is increased fivefold (p < 0.01) in beta cells in type 1 diabetes and in human and rat islets exposed to cytokines that induced increased lactate production. HIF1α attenuates cytokine-induced cell death in beta cells. CONCLUSIONS/INTERPRETATION: The conserved pro-survival HIF1α-mediated injury-response signalling is activated in beta cells in type 1 diabetes and likely contributes to the relatively slow rate of beta cell loss at the expense of early defective glucose-induced insulin secretion.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Secretoras de Insulina/metabolismo , Fosfofrutoquinase-2/metabolismo , Adulto , Idoso de 80 Anos ou mais , Animais , Western Blotting , Linhagem Celular Tumoral , Criança , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imuno-Histoquímica , Imunoprecipitação , Masculino , Fosfofrutoquinase-2/genética , Ratos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Adulto JovemRESUMO
Aberrant mitosis is a common feature of cancer, yet little is known about the altered genes causing mitotic defects. We screened human tumours for cells with morphological signatures of highly specific mitotic defects previously assigned to candidate genes in a genome-wide RNA interference screen carried out in HeLa cells (www.mitocheck.org). We discovered a striking enrichment of early mitotic configurations indicative of prophase/prometaphase delay in breast cancer. Promoter methylation analysis of MitoCheck candidate genes assigned to the corresponding 'mitotic delay' class linked this defect to epigenetic silencing of the gene encoding pregnancy-associated plasma protein-A (PAPPA), a secreted protease. PAPPA silencing was highly prevalent in precursor lesions and invasive breast cancer. Experimental manipulation of PAPPA protein levels in human mammary epithelial cells and in breast cancer cell lines demonstrates that progression through early mitosis is dependent on PAPPA function, and that breast cancer cells become more invasive after down-regulation of this protease. PAPPA regulates mitotic progression through modulating the IGF-1 signalling pathway resulting in activation of the forkhead transcription factor FoxM1, which drives a transcriptional cluster of essential mitotic genes. Our results show that PAPPA has a critical function in normal cell division and is targeted early in breast cancer development.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Epigenômica , Regulação Neoplásica da Expressão Gênica/fisiologia , Inativação Gênica/fisiologia , Mitose/fisiologia , Proteína Plasmática A Associada à Gravidez/fisiologia , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Pessoa de Meia-Idade , Fenótipo , Proteína Plasmática A Associada à Gravidez/genética , Interferência de RNA/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-â£Hdm2-â£p53 â p21 pathway and mediates p15(INK4B) upregulation; p53 in turn activates expression of the Wnt/ß-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Fatores de Transcrição Forkhead/metabolismo , Fase G1/fisiologia , Regulação da Expressão Gênica/genética , Proteínas Serina-Treonina Quinases/metabolismo , Origem de Replicação/genética , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal , Western Blotting , Proteínas de Ciclo Celular/genética , Fracionamento Celular , Linhagem Celular , Quimiocinas , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Primers do DNA/genética , Imunofluorescência , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Humanos , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/cyclosome-Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein-ß-transducin repeat-containing protein (SCF-ß-TrCP), which control the transient appearance and metabolic activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S(273)) within the conserved recognition site for SCF-ß-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-Glu-Asn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-ß-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-to-late G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.
Assuntos
Divisão Celular/fisiologia , Glutaminase/metabolismo , Glicólise/fisiologia , Fosfofrutoquinase-2/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Primers do DNA/genética , Citometria de Fluxo , Glucose/metabolismo , Glutamina/metabolismo , Células HeLa , Humanos , Immunoblotting , Plasmídeos/genética , UbiquitinaçãoRESUMO
During cell proliferation, the abundance of the glycolysis-promoting enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is controlled by the ubiquitin ligase APC/C-Cdh1 via a KEN box. We now demonstrate in synchronized HeLa cells that PFKFB3, which appears in mid-to-late G1, is essential for cell division because its silencing prevents progression into S phase. In cells arrested by glucose deprivation, progression into S phase after replacement of glucose occurs only when PFKFB3 is present or is substituted by the downstream glycolytic enzyme 6-phosphofructo-1-kinase. PFKFB3 ceases to be detectable during late G1/S despite the absence of Cdh1; this disappearance is prevented by proteasomal inhibition. PFKFB3 contains a DSG box and is therefore a potential substrate for SCF-ß-TrCP, a ubiquitin ligase active during S phase. In synchronized HeLa cells transfected with PFKFB3 mutated in the KEN box, the DSG box, or both, we established the breakdown routes of the enzyme at different stages of the cell cycle and the point at which glycolysis is enhanced. Thus, the presence of PFKFB3 is tightly controlled to ensure the up-regulation of glycolysis at a specific point in G1. We suggest that this up-regulation of glycolysis and its associated events represent the nutrient-sensitive restriction point in mammalian cells.
Assuntos
Ciclo Celular/fisiologia , Glicólise/fisiologia , Fosfofrutoquinase-2/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Proliferação de Células , Estabilidade Enzimática , Glucose/metabolismo , Células HeLa , Humanos , Ácido Láctico/metabolismo , Dados de Sequência Molecular , Fosfofrutoquinase-2/genética , Interferência de RNARESUMO
Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteômica , Origem de Replicação , Frações Subcelulares/metabolismo , Linhagem Celular , Cromatografia Líquida , Primers do DNA , Humanos , Interferência de RNA , Espectrometria de Massas em TandemRESUMO
Energy metabolism is a highly conserved process that balances generation of cellular energy and maintenance of redox homeostasis. It consists of five interconnected pathways: glycolysis, tricarboxylic acid cycle, pentose phosphate, trans-sulfuration, and NAD+ biosynthesis pathways. Environmental stress rewires cellular energy metabolism. Type-2 diabetes is a well-studied energy metabolism rewiring state in human pancreatic ß-cells where glucose metabolism is uncoupled from insulin secretion. The two-spotted spider mite, Tetranychus urticae (Koch), exhibits a remarkable ability to adapt to environmental stress. Upon transfer to unfavourable plant hosts, mites experience extreme xenobiotic stress that dramatically affects their survivorship and fecundity. However, within 25 generations, mites adapt to the xenobiotic stress and restore their fitness. Mites' ability to withstand long-term xenobiotic stress raises a question of their energy metabolism states during host adaptation. Here, we compared the transcriptional responses of five energy metabolism pathways between host-adapted and non-adapted mites while using responses in human pancreatic islet donors to model these pathways under stress. We found that non-adapted mites and human pancreatic ß-cells responded in a similar manner to host plant transfer and diabetogenic stress respectively, where redox homeostasis maintenance was favoured over energy generation. Remarkably, we found that upon host-adaptation, mite energy metabolic states were restored to normal. These findings suggest that genes involved in energy metabolism can serve as molecular markers for mite host-adaptation.
Assuntos
Adaptação ao Hospedeiro , Tetranychidae , Animais , Humanos , Tetranychidae/genética , Xenobióticos , Metabolismo EnergéticoRESUMO
HIF1α and PFKFB3 play a critical role in the survival of damaged ß-cells in type-2 diabetes while rendering ß-cells non-responsive to glucose stimulation. To discriminate the role of PFKFB3 from HIF1α in vivo, we generated mice with conditional ß-cell specific disruption of the Pfkfb3 gene on a human islet pancreatic polypeptide (hIAPP+/-) background and a high-fat diet (HFD) [PFKFB3ßKO + diabetogenic stress (DS)]. PFKFB3 disruption in ß-cells under DS led to selective purging of hIAPP-damaged ß-cells and the disappearance of insulin- and glucagon positive bihormonal cells. PFKFB3 disruption induced a three-fold increase in ß-cell replication as evidenced by minichromosome maintenance 2 protein (MCM2) expression. Unlike high-, lower DS or switch to restricted chow diet abolished HIF1α levels and reversed glucose intolerance of PFKFB3ßKO DS mice. Our data suggest that replication and functional recovery of ß-cells under DS depend on ß-cell competitive and selective purification of HIF1α and PFKFB3-positive ß-cells.
Assuntos
Diabetes Mellitus Tipo 2 , Intolerância à Glucose , Células Secretoras de Insulina , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismoRESUMO
Diabetic retinopathy (DR) is the leading cause of blindness for adults in developed countries. Both microvasculopathy and neurodegeneration are implicated in mechanisms of DR development, with neuronal impairment preceding microvascular abnormalities, which is often underappreciated in the clinic. Most current therapeutic strategies, including anti-vascular endothelial growth factor (anti-VEGF)-antibodies, aim at treating the advanced stages (diabetic macular edema and proliferative diabetic retinopathy) and fail to target the neuronal deterioration. Hence, new therapeutic approach(es) intended to address both vascular and neuronal impairment are urgently needed. The hypoxia-inducible factor 1α (HIF1α)-6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) pathway is critically implicated in the islet pathology of diabetes. Recent evidence highlighted the pathway relevance for pathologic angiogenesis and neurodegeneration, two key aspects in DR. PFKFB3 is key to the sprouting angiogenesis, along with VEGF, by determining the endothelial tip-cell competition. Also, PFKFB3-driven glycolysis compromises the antioxidative capacity of neurons leading to neuronal loss and reactive gliosis. Therefore, the HIF1α-PFKFB3 signaling pathway is unique as being a pervasive pathological component across multiple cell types in the retina in the early as well as late stages of DR. A metabolic point-of-intervention based on HIF1α-PFKFB3 targeting thus deserves further consideration in DR.
Assuntos
Retinopatia Diabética/etiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Fosfofrutoquinase-2/fisiologia , Glicólise , Humanos , Mitocôndrias/fisiologia , Neovascularização Patológica/etiologia , Doenças Neurodegenerativas/etiologia , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Retina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
An origin activation checkpoint has recently been discovered in the G1 phase of the mitotic cell cycle, which can be triggered by loss of DNA replication initiation factors such as the Cdc7 kinase. Insufficient levels of Cdc7 activate cell cycle arrest in normal cells, whereas cancer cells appear to lack this checkpoint response, do not arrest, and proceed with an abortive S phase, leading to cell death. The differential response between normal and tumor cells at this checkpoint has led to widespread interest in the development of pharmacological Cdc7 inhibitors as novel anticancer agents. We have used RNAi against Cdc7 in combination with SILAC-based high resolution MS proteomics to investigate the cellular mechanisms underlying the maintenance of the origin activation checkpoint in normal human diploid fibroblasts. Bioinformatics analysis identified clear changes in wide-ranging biological processes including altered cellular energetic flux, moderate stress response, reduced proliferative capacity, and a spatially distributed response across the mitochondria, lysosomes, and the cell surface. These results provide a quantitative overview of the processes involved in maintenance of the arrested state, show that this phenotype involves active rather than passive cellular adaptation, and highlight a diverse set of proteins responsible for cell cycle arrest and ultimately for promotion of cellular survival. We propose that the Cdc7-depleted proteome maintains cellular arrest by initiating a dynamic quiescence-like response and that the complexities of this phenotype will have important implications for the continued development of promising Cdc7-targeted cancer therapies.
Assuntos
Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , Proteômica/métodos , Origem de Replicação/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Humanos , Immunoblotting , Espectrometria de Massas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNARESUMO
PURPOSE: There is a lack of prognostic and predictive biomarkers in epithelial ovarian carcinoma, and the targeting of oncogenic signaling pathways has had limited impact on patient survival in this highly heterogeneous disease. The origin licensing machinery, which renders chromosomes competent for DNA replication, acts as a convergence point for upstream signaling pathways. We tested the hypothesis that Cdc7 kinase, a core component of the licensing machinery, is predictive of clinical outcome and may constitute a novel therapeutic target in epithelial ovarian carcinoma. EXPERIMENTAL DESIGN: A total of 143 cases of ovarian cancer and 5 cases of normal ovary were analyzed for Cdc7 protein expression dynamics and clinicopathologic features. To assess the therapeutic potential of Cdc7, expression was down-regulated by RNA interference in SKOV-3 and Caov-3 ovarian cancer cells. RESULTS: Increased Cdc7 protein levels were significantly associated with arrested tumor differentiation (P = 0.004), advanced clinical stage (P = 0.01), genomic instability (P < 0.001), and accelerated cell cycle progression. Multivariate analysis shows that Cdc7 predicts disease-free survival independent of patient age, tumor grade and stage (hazard ratio, 2.03; confidence interval, 1.53-2.68; P < 0.001), with the hazard ratio for relapse increasing to 10.90 (confidence interval, 4.07-29.17) for the stages 3 to 4/upper Cdc7 tertile group relative to stages 1 to 2/lower Cdc7 tertile tumors. In SKOV-3 and Caov-3 cells, Cdc7 siRNA knockdown triggered high levels of apoptosis, whereas untransformed cells arrest in G(1) phase and remain viable. CONCLUSIONS: Our findings show that Cdc7 kinase predicts survival and is a potent anticancer target in epithelial ovarian carcinoma, highlighting its potential as a predictor of susceptibility to small molecule kinase inhibitors currently in development.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma/mortalidade , Proteínas de Ciclo Celular/metabolismo , Neoplasias Ovarianas/mortalidade , Proteínas Serina-Treonina Quinases/metabolismo , Apoptose , Biomarcadores Tumorais/análise , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Instabilidade Genômica , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Ovário/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Análise de SobrevidaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The islet in type 2 diabetes (T2D) is characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by ß-cells. In common with amyloidogenic proteins implicated in neurodegeneration, human IAPP (hIAPP) forms membrane permeant toxic oligomers implicated in misfolded protein stress. Here, we establish that hIAPP misfolded protein stress activates HIF1α/PFKFB3 signaling, this increases glycolysis disengaged from oxidative phosphorylation with mitochondrial fragmentation and perinuclear clustering, considered a protective posture against increased cytosolic Ca2+ characteristic of toxic oligomer stress. In contrast to tissues with the capacity to regenerate, ß-cells in adult humans are minimally replicative, and therefore fail to execute the second pro-regenerative phase of the HIF1α/PFKFB3 injury pathway. Instead, ß-cells in T2D remain trapped in the pro-survival first phase of the HIF1α injury repair response with metabolism and the mitochondrial network adapted to slow the rate of cell attrition at the expense of ß-cell function.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Estresse do Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Adulto , Animais , Animais Geneticamente Modificados , Apoptose , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Glicólise/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Masculino , Pessoa de Meia-Idade , Mitofagia/fisiologia , Fosforilação Oxidativa , Fosfofrutoquinase-2/metabolismo , Agregados Proteicos/fisiologia , RatosRESUMO
Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca2+ are critical for permitting cells to progress through the cell cycle. Here, we investigated these parameters in the replication-competent beta-cell line INS 832/13. Cell cycle synchronization of this line permitted evaluation of cell metabolism, mitochondrial network, and cellular Ca2+ compartmentalization at key cell cycle stages. The mitochondrial network is interconnected and filamentous at G1/S but fragments during the S and G2/M phases, presumably to permit sorting to daughter cells. Pyruvate anaplerosis peaks at G1/S, consistent with generation of biomass for daughter cells, whereas mitochondrial Ca2+ and respiration increase during S and G2/M, consistent with increased energy requirements for DNA and lipid synthesis. This synchronization approach may be of value to investigators performing live cell imaging of Ca2+ or mitochondrial dynamics commonly undertaken in INS cell lines because without synchrony widely disparate data from cell to cell would be expected depending on position within cell cycle. Our findings also offer insight into why replicating beta-cells are relatively nonfunctional secreting insulin in response to glucose. They also provide guidance on metabolic requirements of beta-cells for the transition through the cell cycle that may complement the efforts currently restricted to manipulating cell cycle to drive beta-cells through cell cycle.
Assuntos
Ciclo Celular/fisiologia , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Divisão Celular/fisiologia , Linhagem Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Replicação do DNA/genética , Mitocôndrias/genética , RatosRESUMO
DNA replication initiation is a key event in the cell cycle, which is dependent on 2 kinases - CDK2 and CDC7. Here we report a novel mechanism in which p53 induces G1 checkpoint and cell cycle arrest by downregulating CDC7 kinase in response to genotoxic stress. We demonstrate that p53 controls CDC7 stability post-transcriptionally via miR-192/215 and post-translationally via Fbxw7ß E3 ubiquitin ligase. The p53-dependent pathway of CDC7 downregulation is interlinked with the p53-p21-CDK2 pathway, as p21-mediated inhibition of CDK2-dependent phosphorylation of CDC7 on Thr376 is required for GSK3ß-phosphorylation and Fbxw7ß-dependent degradation of CDC7. Notably, sustained oncogenic high levels of active CDC7 exert a negative feedback onto p53, leading to unrestrained S-phase progression and accumulation of DNA damage. Thus, p53-dependent control of CDC7 levels is essential for blocking G1/S cell-cycle transition upon genotoxic stress, thereby safeguarding the genome from instability and thus representing a novel general stress response.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Pontos de Checagem da Fase G1 do Ciclo Celular , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo/genética , Retroalimentação Fisiológica , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HCT116 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Ubiquitina-Proteína Ligases/metabolismoRESUMO
PURPOSE: Cdc7 is a serine/threonine kinase which is responsible for the 'firing' of replication origins leading to initiation of DNA replication. Inhibition or depletion of Cdc7 in normal cells triggers a DNA origin activation checkpoint causing a reversible G1 arrest. Here we investigate Cdc7 as a novel therapeutic target in pancreatic cancer. EXPERIMENTAL DESIGN: Cdc7 target validation was performed by immunoexpression profiling in a cohort of 73 patients with pancreatic adenocarcinoma including 24 controls. Secondly Cdc7 kinase was targeted in Capan-1 and PANC-1 pancreatic cancer cell line models using either an siRNA against Cdc7 or alternatively a small molecule inhibitor (SMI) of Cdc7 (PHA-767491). RESULTS: Cdc7 was significantly overexpressed in pancreatic adenocarcinoma compared to benign pancreatic tissue (median LI 34.3% vs. 1.3%; P<0.0001). Cdc7 knockdown using siRNA in Capan-1 and PANC-1 cells resulted in marked apoptotic cell death when compared with control cells. A prominent sub-G1 peak was seen on flow cytometry (sub-G1 51% vs. 3% and 45% vs. 0.7% in Capan-1 and PANC-1 cells, respectively). Annexin V labelling confirmed apoptosis in 64% vs. 11% and 75% vs. 8%, respectively. Western blotting showed cleavage of PARP-1 and caspase-3 and presence of γH2A.X. TUNEL assay showed strong staining in treated cells. These results were mirrored following Cdc7 kinase inhibition with PHA-767491. CONCLUSIONS: Our findings show that Cdc7 is a potent anti-cancer target in pancreatic adenocarcinoma and that Cdc7 immunoexpression levels might be used as a companion diagnostic to predict response to therapeutic siRNAs or SMIs directed against this kinase.
Assuntos
Adenocarcinoma/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Replicação do DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Piperidonas/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirróis/farmacologia , TransfecçãoRESUMO
The recent recognition of the clinical association between type 2 diabetes (T2D) and several types of human cancer has been further highlighted by reports of antidiabetic drugs treating or promoting cancer. At the cellular level, a plethora of molecules operating within distinct signaling pathways suggests cross-talk between the multiple pathways at the interface of the diabetes-cancer link. Additionally, a growing body of emerging evidence implicates homeostatic pathways that may become imbalanced during the pathogenesis of T2D or cancer or that become chronically deregulated by prolonged drug administration, leading to the development of cancer in diabetes and vice versa. This notion underscores the importance of combining clinical and basic mechanistic studies not only to unravel mechanisms of disease development but also to understand mechanisms of drug action. In turn, this may help the development of personalized strategies in which drug doses and administration durations are tailored to individual cases at different stages of the disease progression to achieve more efficacious treatments that undermine the diabetes-cancer association.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Neoplasias/metabolismo , Animais , Diabetes Mellitus Tipo 2/terapia , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias/terapia , Via de Sinalização WntRESUMO
The small molecule carrier class of biomolecule transporters, modeled on the third helix of the Antennapedia homeodomain, has previously been shown to transport active proteins into cells. Here, we show an improved synthetic route to small molecule carriers, including Molander chemistry using trifluoroborate salts to improve the yield of the Suzuki-Miyaura coupling step for the formation of the biphenyl backbone. The required boronic acids could be formed by the reaction of a 2-(dimethylamino)ethyl ether-modified aryl Grignard reagent with triisopropyl borate. The potential for the use of small molecule carriers as oligonucleotide-transporting agents was also explored by characterizing the interactions between small molecule carriers and siRNA. Molecular dynamics and NMR analysis indicated that the small molecule carrier guanidines are stabilized by π-cation interactions with the biphenyl system, thus not only increasing the basicity or pKa but also shielding the charge. The binding affinities of various small molecule carriers for siRNA were investigated using isothermal calorimetry and gel shift assays. Small molecule carrier-mediated siRNA delivery to cultured fibroblasts is demonstrated, showing that small molecule carriers possess the ability to transport functional siRNA into cells. Knockdown of Cdc7 kinase, a target for cancer, is achieved.