RESUMEN

Oral squamous cell carcinoma (OSCC) is still an unabated global killer with little advancement in its survival rate. DNA replication licensing proteins and Aurora kinase A are biomarkers that play important roles in genomic stability. The expression profile of minichromosomal maintenance protein 2 (MCM2), Ki67, geminin, and Aurora-A were linked to clinicopathological and outcome parameters, survival, and DNA content in 125 cases of OSCC. Oral fibroepithelial polyps (OFEP) were controls. The OSCC tumour cells were in a rapidly proliferating state, as assessed by the increased expression profile of MCM2, Ki67, geminin, and Aurora-A and of the geminin/Ki67 ratio, and the decrease of the MCM2/Ki67 ratio, in OSCC compared with OFEP (P < 0.000). There was an association between expression of MCM2, Ki67, and geminin and tumour histologic and invasive front grade (P < 0.05). A total of 82% of the OSCC assessed had aneuploid DNA content, which was associated with increased expression intensity of Aurora-A (P = 0.01). Geminin and the geminin/Ki67 ratio were associated with TNM staging (P < 0.05), and weak expression of MCM2, Ki67, geminin, and Aurora-A were predictive of OSCC survival (P < 0.05). Dysregulation of the origin licensing pathway and the mitotic pathway are important events in OSCC, and the combined analysis of these proteins may contribute to improved treatment decisions.

Asunto(s)

Carcinoma de Células Escamosas/metabolismo , Geminina/metabolismo , Antígeno Ki-67/metabolismo , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Neoplasias de la Boca/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Aurora Quinasa A/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Proliferación Celular/fisiología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología

RESUMEN

Cell cycle status may play an important role in directing patient therapy. We therefore determined the cell cycle status of leukaemic cells by immunophenotypic analysis of bone marrow trephine biopsies from 181 patients with acute myeloid leukaemia (AML) and correlated the results with biological features and clinical outcome. There was considerable heterogeneity between patients. The presenting white cell count significantly correlated with the proportion of non-quiescent cells (P < 0·0001), of cycling cells beyond G1 (P < 0·0001) and the speed of cycling (P < 0·0001). Profiles in acute promyelocytic leukaemia (APL) differed from non-APL and were consistent with more differentiated cells with reduced proliferative potential, but no significant differences were observed between non-APL cytogenetic risk groups. NPM1 mutations but not FLT3 internal tandem duplication (FLT3ITD ) were significantly associated with a higher proportion of cells beyond G1 (P = 0·002) and faster speed of cycling (P = 0·003). Resistance to standard cytosine arabinoside and daunorubicin induction chemotherapy was significantly related to a slower speed of cycling (P = 0·0002), as was a higher relapse rate (P = 0·05), but not with the proportion of non-quiescent cells or actively cycling cells. These results show a link between the cycling speed of AML cells and the response to chemotherapy, and help to identify a group with a very poor prognosis.

Asunto(s)

Ciclo Celular , Genotipo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Mutación , Proteínas Nucleares , Tirosina Quinasa 3 Similar a fms , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Citarabina/farmacología , Daunorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Inmunofenotipificación , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/patología , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo

RESUMEN

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.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Puntos de Control de la Fase G1 del Ciclo Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Ciclo Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo/genética , Retroalimentación Fisiológica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HCT116 , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

MS-based proteomics has been applied to a differential network analysis of the nuclear-cytoplasmic subcellular distribution of proteins between cell-cycle arrest: (a) at the origin activation checkpoint for DNA replication, or (b) in response to oxidative stress. Significant changes were identified for 401 proteins. Cellular response combines changes in trafficking and in total abundance to vary the local compartmental abundances that are the basis of cellular response. Appreciable changes for both perturbations were observed for 245 proteins, but cross-talk between oxidative stress and DNA replication is dominated by 49 proteins that show strong changes for both. Many nuclear processes are influenced by a spatial switch involving the proteins {KPNA2, KPNB1, PCNA, PTMA, SET} and heme/iron proteins HMOX1 and FTH1. Dynamic spatial distribution data are presented for proteins involved in caveolae, extracellular matrix remodelling, TGFß signaling, IGF pathways, emerin complexes, mitochondrial protein import complexes, spliceosomes, proteasomes, and so on. The data indicate that for spatially heterogeneous cells cross-compartmental communication is integral to their system biology, that coordinated spatial redistribution for crucial protein networks underlies many functional changes, and that information on dynamic spatial redistribution of proteins is essential to obtain comprehensive pictures of cellular function. We describe how spatial data of the type presented here can provide priorities for further investigation of crucial features of high-level spatial coordination across cells. We suggest that the present data are related to increasing indications that much of subcellular protein transport is constitutive and that perturbation of these constitutive transport processes may be related to cancer and other diseases. A quantitative, spatially resolved nucleus-cytoplasm interaction network is provided for further investigations.

Asunto(s)

Compartimento Celular , Replicación del ADN , Fibroblastos/química , Estrés Oxidativo , Proteoma/análisis , Fracciones Subcelulares/química , Puntos de Control del Ciclo Celular , Línea Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Fibroblastos/citología , Fibroblastos/ultraestructura , Humanos , Transporte de Proteínas , Proteómica/métodos , Fracciones Subcelulares/metabolismo

RESUMEN

Using immunohistochemistry and flow cytometry to define phases of the cell cycle, this study shows that a high proportion of acute myeloid leukaemia (AML) blasts obtained from trephine biopsies are cycling, whereas >95% of peripheral blood-derived blasts are arrested in G1 . Results obtained from bone marrow aspirates are more similar to those from blood rather than from trephine biopsies. These differences were confirmed by gene expression profiling in a patient with high count AML. This has implications for cell cycle and other biological studies using aspirates rather than trephine biopsies and for the use of cell mobilising agents before chemotherapy.

Asunto(s)

Crisis Blástica/patología , Ciclo Celular , Leucemia Mieloide Aguda/patología , Adulto , Anciano , Biopsia , Células de la Médula Ósea/patología , Puntos de Control del Ciclo Celular , Femenino , Fase G1 , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Células Neoplásicas Circulantes/patología , Trepanación

RESUMEN

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.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Estudios de Cohortes , Replicación del ADN , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Humanos , Terapia Molecular Dirigida , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Piperidonas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pirroles/farmacología , Transfección

RESUMEN

We have used a subcellular spatial razor approach based on LC-MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function.

Asunto(s)

Núcleo Celular/metabolismo , Citoplasma/metabolismo , Estrés Oxidativo , Línea Celular , Cromatografía Liquida , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Transporte de Proteínas , Espectrometría de Masas en Tándem

RESUMEN

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.

Asunto(s)

Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Epigenómica , Regulación Neoplásica de la Expresión Génica/fisiología , Silenciador del Gen/fisiología , Mitosis/fisiología , Proteína Plasmática A Asociada al Embarazo/fisiología , Anciano , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Persona de Mediana Edad , Fenotipo , Proteína Plasmática A Asociada al Embarazo/genética , Interferencia de ARN/fisiología , Transducción de Señal/fisiología

RESUMEN

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.

Asunto(s)

Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteómica , Origen de Réplica , Fracciones Subcelulares/metabolismo , Línea Celular , Cromatografía Liquida , Cartilla de ADN , Humanos , Interferencia de ARN , Espectrometría de Masas en Tándem

RESUMEN

BACKGROUND: Urinary biomarkers for bladder cancer detection are constrained by inadequate sensitivity or specificity. Here we evaluate the diagnostic accuracy of Mcm5, a novel cell cycle biomarker of aberrant growth, alone and in combination with NMP22. METHODS: 1677 consecutive patients under investigation for urinary tract malignancy were recruited to a prospective blinded observational study. All patients underwent ultrasound, intravenous urography, cystoscopy, urine culture and cytologic analysis. An immunofluorometric assay was used to measure Mcm5 levels in urine cell sediments. NMP22 urinary levels were determined with the FDA-approved NMP22® Test Kit. RESULTS: Genito-urinary tract cancers were identified in 210/1564 (13%) patients with an Mcm5 result and in 195/1396 (14%) patients with an NMP22 result. At the assay cut-point where sensitivity and specificity were equal, the Mcm5 test detected primary and recurrent bladder cancers with 69% sensitivity (95% confidence interval = 62-75%) and 93% negative predictive value (95% CI = 92-95%). The area under the receiver operating characteristic curve for Mcm5 was 0.75 (95% CI = 0.71-0.79) and 0.72 (95% CI = 0.67-0.77) for NMP22. Importantly, Mcm5 combined with NMP22 identified 95% (79/83; 95% CI = 88-99%) of potentially life threatening diagnoses (i.e. grade 3 or carcinoma in situ or stage ≥pT1) with high specificity (72%, 95% CI = 69-74%). CONCLUSIONS: The Mcm5 immunoassay is a non-invasive test for identifying patients with urothelial cancers with similar accuracy to the FDA-approved NMP22 ELISA Test Kit. The combination of Mcm5 plus NMP22 improves the detection of UCC and identifies 95% of clinically significant disease. Trials of a commercially developed Mcm5 assay suitable for an end-user laboratory alongside NMP22 are required to assess their potential clinical utility in improving diagnostic and surveillance care pathways.

Asunto(s)

Biomarcadores de Tumor/orina , Carcinoma de Células Transicionales/diagnóstico , Proteínas de Ciclo Celular/orina , Proteínas Nucleares/orina , Neoplasias de la Vejiga Urinaria/diagnóstico , Anciano , Área Bajo la Curva , Carcinoma , Carcinoma de Células Transicionales/orina , Reacciones Falso Positivas , Femenino , Humanos , Límite de Detección , Masculino , Persona de Mediana Edad , Curva ROC , Estadísticas no Paramétricas , Neoplasias de la Vejiga Urinaria/orina

RESUMEN

Deregulation of the cell cycle underlies the aberrant cell proliferation that characterizes cancer and loss of cell cycle checkpoint control promotes genetic instability. During the past two decades, cancer genetics has shown that hyperactivating mutations in growth signalling networks, coupled to loss of function of tumour suppressor proteins, drives oncogenic proliferation. Gene expression profiling of these complex and redundant mitogenic pathways to identify prognostic and predictive signatures and their therapeutic targeting has, however, proved challenging. The cell cycle machinery, which acts as an integration point for information transduced through upstream signalling networks, represents an alternative target for diagnostic and therapeutic interventions. Analysis of the DNA replication initiation machinery and mitotic engine proteins in human tissues is now leading to the identification of novel biomarkers for cancer detection and prognostication, and is providing target validation for cell cycle-directed therapies.

Asunto(s)

Ciclo Celular/fisiología , Neoplasias/patología , Antineoplásicos/uso terapéutico , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/efectos de los fármacos , Replicación del ADN/fisiología , Detección Precoz del Cáncer , Geminina , Humanos , Neoplasias/tratamiento farmacológico , Fosfoproteínas Fosfatasas/metabolismo , Fosfotransferasas/metabolismo , Transducción de Señal/fisiología , Resultado del Tratamiento

RESUMEN

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.

Asunto(s)

ARN Interferente Pequeño/química , Bibliotecas de Moléculas Pequeñas/química , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Guanidina/química , Humanos , Cinética , Microscopía Confocal , Simulación de Dinámica Molecular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química

RESUMEN

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.

Asunto(s)

Ciclo Celular/fisiología , Glucólisis/fisiología , Fosfofructoquinasa-2/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Proliferación Celular , Estabilidad de Enzimas , Glucosa/metabolismo , Células HeLa , Humanos , Ácido Láctico/metabolismo , Datos de Secuencia Molecular , Fosfofructoquinasa-2/genética , Interferencia de ARN

RESUMEN

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.

Asunto(s)

Ciclo Celular/fisiología , Replicación del ADN/fisiología , Proteómica/métodos , Origen de Réplica/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Humanos , Immunoblotting , Espectrometría de Masas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN

RESUMEN

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-∣Hdm2-∣p53 → 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.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/genética , Factores de Transcripción Forkhead/metabolismo , Fase G1/fisiología , Regulación de la Expresión Génica/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Origen de Réplica/genética , Transducción de Señal/genética , Proteínas Adaptadoras Transductoras de Señales , Western Blotting , Proteínas de Ciclo Celular/genética , Fraccionamiento Celular , Línea Celular , Quimiocinas , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Humanos , Immunoblotting , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Treatment options for triple-receptor negative (ER-/PR-/Her2-) and Her2-overexpressing (ER-/PR-/Her2+) breast cancers with acquired or de novo resistance are limited, and metastatic disease remains incurable. Targeting of growth signaling networks is often constrained by pathway redundancy or growth-independent cancer cell cycles. The cell-cycle protein Cdc7 regulates S phase by promoting DNA replication. This essential kinase acts as a convergence point for upstream growth signaling pathways and is therefore an attractive therapeutic target. We show that increased Cdc7 expression during mammary tumorigenesis is linked to Her2-overexpressing and triple-negative subtypes, accelerated cell cycle progression (P < 0.001), arrested tumor differentiation (P < 0.001), genomic instability (P = 0.019), increasing NPI score (P < 0.001), and reduced disease-free survival (HR = 1.98 [95% CI: 1.27-3.10]; P = 0.003), thus implicating its deregulation in the development of aggressive disease. Targeting Cdc7 with RNAi, we demonstrate that p53-mutant Her2-overexpressing and triple-negative breast cancer cell lines undergo an abortive S phase and apoptotic cell death due to loss of a p53-dependent Cdc7-inhibition checkpoint. In contrast, untransformed breast epithelial cells arrest in G1, remain viable, and are able to resume cell proliferation on recovery of Cdc7 kinase activity. Thus, Cdc7 appears to represent a potent and highly specific anticancer target in Her2-overexpressing and triple-negative breast cancers. Emerging Cdc7 kinase inhibitors may therefore significantly broaden the therapeutic armamentarium for treatment of the aggressive p53-mutant breast cancer subtypes identified in this study.

Asunto(s)

Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Genes p53/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S/fisiología , Apoptosis , Western Blotting , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , Femenino , Humanos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal

RESUMEN

Cancer biomarkers provide an opportunity to diagnose tumours earlier and with greater accuracy. They can also identify those patients most at risk of disease recurrence and predict which tumours will respond to different therapeutic approaches. Such biomarkers will be especially useful in the diagnosis and management of bladder cancer. At present, bladder tumours are diagnosed and followed-up using a combination of cystoscopic examination, cytology and histology. These are not only expensive, but also highly subjective investigations and reveal little about the underlying molecular characteristics of the tumour. In recent years numerous diagnostic and prognostic biomarkers of bladder cancer have been identified. Two separate approaches to biomarker discovery have been employed. The first is hypothesis-driven and focuses upon proteins involved in molecular pathways known to be implicated in tumorigenesis. An alternative approach has been to study the global expression of genes (so-called 'genomics') looking for characteristic signatures associated with disease outcomes. In this review we summarize the current state of biomarker development in this field, and examine why so few have made the successful transition into the clinic. Finally, we introduce a novel approach to biomarker development utilizing components of the DNA replication licensing machinery.

Asunto(s)

Biomarcadores de Tumor/análisis , Neoplasias de la Vejiga Urinaria/química , Neoplasias de la Vejiga Urinaria/diagnóstico , Algoritmos , Antígenos de Neoplasias/orina , Apoptosis , Biomarcadores de Tumor/orina , Ciclo Celular , Proteínas de Ciclo Celular/análisis , Ciclinas/análisis , Citodiagnóstico/métodos , Replicación del ADN , Perfilación de la Expresión Génica , Genes p53 , Humanos , Hibridación Fluorescente in Situ , Antígeno Ki-67/análisis , Sistema de Señalización de MAP Quinasas/genética , Proteínas Nucleares/orina , Pronóstico , Neoplasias de la Vejiga Urinaria/genética

RESUMEN

PURPOSE: The DNA replication licensing machinery is integral to the control of proliferation, differentiation, and maintenance of genomic stability in human cells. We have analyzed replication licensing factors (RLF), together with DNA ploidy status, to investigate their role in progression of penile squamous cell carcinoma and to assess their utility as novel prognostic tools. EXPERIMENTAL DESIGN: In a cohort of 141 patients, we linked protein expression profiles of the standard proliferation marker Ki67 and the RLFs Mcm2 and geminin to clinicopathologic variables, ploidy status, and clinical outcome. RESULTS: Increased Ki67, Mcm2, and geminin levels were each significantly associated with arrested tumor differentiation (P < 0.0001) and aneuploidy (P < or = 0.01). Accelerated cell cycle progression was linked to increasing tumor size, stage, and depth of invasion. Aneuploid tumors significantly correlated with tumor grade (P < 0.0001). Biomarker expression and DNA ploidy status were significant predictors of locoregional disease progression [Mcm2 (P = 0.02), geminin (P = 0.02), Ki67 (P = 0.03), and aneuploidy (P = 0.03)] in univariate analysis. Importantly, aneuploidy was a strong independent prognosticator for overall survival (hazard ratio, 4.19; 95% confidence interval, 1.17-14.95; P = 0.03). Used in conjunction with conventional pathologic information, multiparameter analysis of these variables can stratify patients into low- or high-risk groups for disease progression (Harrell's c-index = 0.88). CONCLUSIONS: Our findings suggest that RLFs and tumor aneuploidy may be used as an adjunct to conventional prognostic indicators, identifying men at high risk of disease progression. Our results also identify the DNA replication initiation pathway as a potentially attractive therapeutic target in penile squamous cell carcinoma.

Asunto(s)

Aneuploidia , Carcinoma/genética , Carcinoma/terapia , Regulación Neoplásica de la Expresión Génica , Neoplasias del Pene/genética , Neoplasias del Pene/terapia , Adulto , Anciano , Anciano de 80 o más Años , Ciclo Celular , Proteínas de Ciclo Celular/biosíntesis , Estudios de Cohortes , Geminina , Perfilación de la Expresión Génica , Humanos , Antígeno Ki-67/biosíntesis , Masculino , Persona de Mediana Edad , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Proteínas Nucleares/biosíntesis , Ploidias , Resultado del Tratamiento

RESUMEN

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.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Carcinoma/mortalidad , Proteínas de Ciclo Celular/metabolismo , Neoplasias Ováricas/mortalidad , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis , Biomarcadores de Tumor/análisis , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Células Cultivadas , Femenino , Inestabilidad Genómica , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Ovario/enzimología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Análisis de Supervivencia

RESUMEN

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

Asunto(s)

Secuencias de Aminoácidos , Arginina/química , Replicación del ADN , Mupapillomavirus/patogenicidad , Proteínas de Fusión Oncogénica/química , Proteínas Oncogénicas Virales/fisiología , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , ADN/biosíntesis , Células HeLa , Humanos , Ratones , Mupapillomavirus/metabolismo , Células 3T3 NIH , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Fase S , Spodoptera , Proteínas Virales/química , Xenopus laevis