RESUMEN
Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.
Asunto(s)
Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/genética , Leucemia/tratamiento farmacológico , Metiltransferasas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Animales , Apoptosis , Proliferación Celular , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Leucémica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Leucemia/genética , Leucemia/patología , Metiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Células Tumorales CultivadasRESUMEN
Here we report that promoter mutations in telomerase (TERT), the most common noncoding mutations in cancer, give rise to monoallelic expression of TERT. Through deep RNA sequencing, we find that TERT activation in human cancer cell lines can occur in either mono- or biallelic manner. Without exception, hotspot TERT promoter mutations lead to the re-expression of only one allele, accounting for approximately half of the observed cases of monoallelic TERT expression. Furthermore, we show that monoallelic TERT expression is highly prevalent in certain tumor types and widespread across a broad spectrum of cancers. Taken together, these observations provide insights into the mechanisms of TERT activation and the ramifications of noncoding mutations in cancer.
RESUMEN
An increased understanding of the molecular etiology of cancer has enabled the development of novel therapies that are collectively referred to as molecular targeted agents. Unlike the drugs used in conventional chemotherapy, these agents are designed to specifically interfere with key molecular events that are responsible for the malignant phenotype. They hold great promise for widening the therapeutic window, which would provide more effective treatment options as compared with cytotoxic therapies. In addition, the targeted approach that is characteristic of these drugs provides unique opportunities for combination therapies with other anticancer agents that have non-overlapping toxicities. Targeted agents are therefore primed to become invaluable therapeutic tools in the multimodal treatment of cancer. The challenges associated with these novel targeted therapies are distinct from those faced in conventional chemotherapy. These unique challenges include the need to select appropriate pharmacodynamic markers to guide dose and schedule and to identify biomarkers that enable selection of patient populations that are most likely to benefit from the treatment. In addition, although the emergence of resistance to targeted therapies is a problem frequently faced in the clinic, the molecular characterization of resistance mechanisms provides the opportunity to design second-generation therapies or combination therapies aimed at preventing resistance or restoring response. The development of the tyrosine kinase inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia (CML). In this article, we discuss the lessons learned from the application of imatinib and other targeted agents in clinical practice and discuss how these insights may guide the development of novel targeted therapies.
Asunto(s)
Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos/tendencias , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificación , Animales , Antineoplásicos/uso terapéutico , Benzamidas , Sistemas de Liberación de Medicamentos/métodos , Quimioterapia Combinada , Humanos , Mesilato de Imatinib , Neoplasias/genética , Neoplasias/mortalidad , Piperazinas/uso terapéutico , Pirimidinas/uso terapéuticoRESUMEN
Although 75% of endometrial cancers are treated at an early stage, 15% to 20% of these recur. We performed an integrated analysis of genome-wide expression and copy-number data for primary endometrial carcinomas with extensive clinical and histopathological data to detect features predictive of recurrent disease. Unsupervised analysis of the expression data distinguished 2 major clusters with strikingly different phenotypes, including significant differences in disease-free survival. To identify possible mechanisms for these differences, we performed a global genomic survey of amplifications, deletions, and loss of heterozygosity, which identified 11 significantly amplified and 13 significantly deleted regions. Amplifications of 3q26.32 harboring the oncogene PIK3CA were associated with poor prognosis and segregated with the aggressive transcriptional cluster. Moreover, samples with PIK3CA amplification carried signatures associated with in vitro activation of PI3 kinase (PI3K), a signature that was shared by aggressive tumors without PIK3CA amplification. Tumors with loss of PTEN expression or PIK3CA overexpression that did not have PIK3CA amplification also shared the PI3K activation signature, high protein expression of the PI3K pathway member STMN1, and an aggressive phenotype in test and validation datasets. However, mutations of PTEN or PIK3CA were not associated with the same expression profile or aggressive phenotype. STMN1 expression had independent prognostic value. The results affirm the utility of systematic characterization of the cancer genome in clinically annotated specimens and suggest the particular importance of the PI3K pathway in patients who have aggressive endometrial cancer.
Asunto(s)
Neoplasias Endometriales/enzimología , Neoplasias Endometriales/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genoma Humano/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Biomarcadores de Tumor/metabolismo , Fosfatidilinositol 3-Quinasa Clase I , Análisis por Conglomerados , Neoplasias Endometriales/diagnóstico , Neoplasias Endometriales/patología , Activación Enzimática , Femenino , Dosificación de Gen , Humanos , Pérdida de Heterocigocidad/genética , Pronóstico , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Estatmina/metabolismo , Análisis de Supervivencia , Proteínas ras/metabolismoRESUMEN
The abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been validated by epidemiological and experimental studies as an essential step toward the initiation and maintenance of human tumors. Notable in this regard are the prevalent somatic genetic alterations leading to the inactivation of the tumor suppressor gene PTEN and gain-of-function mutations targeting PIK3CA--the gene encoding the catalytic phosphosinositide-3 kinase subunit p110 alpha. A number of the intracellular components of this pathway have been targeted as anticancer drug discovery activities leading to the current panoply of clinical trials of inhibitors of PI3K, Akt and HSP90 in man. This review summarizes current preclinical knowledge of modulators of the PI3K/Akt pathway in which drug discovery and development activities have been advanced focusing on both the relevant clinical stage inhibitors and other disclosed tool compounds targeting PI3K, PDK1, Akt and HSP90.
Asunto(s)
Antineoplásicos/síntesis química , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Antineoplásicos/análisis , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Genome-era advances in the field of oncology endorse the notion that many tumors may prove vulnerable to targeted therapeutic avenues once their salient molecular alterations are elucidated. Accomplishing this requires both detailed genomic characterization and the ability to identify in situ the critical dependencies operant within individual tumors. To this end, DNA microarray platforms such as high-density single-nucleotide polymorphism (SNP) arrays enable large-scale cancer genome characterization, including copy number and loss-of-heterozygosity analyses at high resolution. Clustering analyses of SNP array data from a large collection of tumor samples and cell lines suggest that certain copy number alterations correlate strongly with the tissue of origin. Such lineage-restricted alterations may harbor novel cancer genes directing genesis or progression of tumors from distinct tissue types. We have explored this notion through combined analysis of genome-scale data sets from the NCI60 cancer cell line collection. Here, several melanoma cell lines clustered on the basis of increased dosage at a region of chromosome 3p containing the master melanocyte regulator MITF. Combined analysis of gene expression data and additional functional studies established MITF as an amplified oncogene in melanoma. MITF may therefore represent a nodal point within a critical lineage survival pathway operant in a subset of melanomas. These findings suggest that, like oncogene addiction, "lineage addiction" may represent a fundamental tumor survival mechanism with important therapeutic implications.
Asunto(s)
Genómica/métodos , Neoplasias/genética , Cromosomas Humanos Par 3/genética , Análisis por Conglomerados , ADN de Neoplasias/genética , Amplificación de Genes , Dosificación de Gen , Perfilación de la Expresión Génica , Genómica/estadística & datos numéricos , Humanos , Hibridación Fluorescente in Situ , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oncogenes , Polimorfismo de Nucleótido SimpleRESUMEN
The complete sequencing of the human genome and the development of molecularly targeted cancer therapy have promoted efforts to identify systematically the genetic alterations in human cancer. By high-throughput sequencing of tyrosine kinase genes in human non-small-cell lung cancer, we identified somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene (EGFR) that are correlated with clinical response to EGFR tyrosine kinase inhibitors (TKIs). We have shown that these mutant forms of EGFR induce oncogenic transformation in different cellular systems. Cells whose growth depends on EGFR with mutations in exons 19 and 21 are sensitive to EGFR-TKIs, whereas cells expressing insertion mutations in exon 20 or the T790M point mutant, found in tumor biopsies from patients that relapsed after an initial response to EGFR-TKIs, are resistant. Furthermore, by applying a novel, massively parallel sequencing technology, we have shown that clinically relevant oncogene mutations can be detected in clinical specimens with very low tumor content, thereby enabling optimal patient selection for mutation-directed therapy. In summary, by applying high-throughput genomic resequencing, we have identified a novel therapeutic target, mutant EGFR, in lung cancer and evaluated its role in predicting response to targeted therapy.
Asunto(s)
Adenocarcinoma/genética , Genes erbB-1 , Neoplasias Pulmonares/genética , Mutación , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/enzimología , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Receptores ErbB/química , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Ratones , Células 3T3 NIH , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de ProteínaRESUMEN
PTEN is a tumor suppressor protein that functions, in large part, by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate and by doing so antagonizing the action of phosphoinositide 3-kinase. PTEN structural domains include an N-terminal phosphatase domain, a lipid-binding C2 domain, and a 50-amino acid C-terminal tail that contains a PDZ binding sequence. We showed previously that phosphorylation of the PTEN tail negatively regulates PTEN activity. We now show that phosphorylated PTEN exists in a monomeric "closed" conformation and has low affinity for PDZ domain-containing proteins. Conversely, when unphosphorylated, PTEN is in an "open" conformation, is recruited into a high molecular weight complex (PTEN-associated complex), and strongly interacts with PDZ-containing proteins such as MAGI-2. As a consequence, when compared with wild-type PTEN, the phosphorylation-deficient mutant form of PTEN strongly cooperates with MAGI-2 to block Akt activation. These results indicate that phosphorylation of the PTEN tail causes a conformational change that results in the masking of the PDZ binding domain. Consequently, the ability of PTEN to bind to PDZ domain-containing proteins is reduced dramatically. These data suggest that phosphorylation of the PTEN tail suppresses the activity of PTEN by controlling the recruitment of PTEN into the PTEN-associated complex.
Asunto(s)
Receptores de Activinas Tipo II , Proteínas Portadoras/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Línea Celular , Guanilato-Quinasas , Humanos , Isoenzimas , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/química , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Proteínas Supresoras de Tumor/químicaRESUMEN
The retinoblastoma tumor suppressor protein has been shown to bind directly and inhibit a transcriptionally-important amino-terminal kinase domain of TATA-binding protein-associated factor TAFII250. Cyclin D1 also is able to associate with the amino terminus of TAFII250 in a region very similar to or overlapping the Rb-binding site. In this study, we have examined whether cyclin D1 affects the functional interaction between Rb and TAFII250. We observed that when cyclin D1 is coincubated with Rb and TAFII250, the ability of Rb to inhibit TAFII250 kinase activity is effectively blocked. However, cyclin D1 by itself has no apparent effect on TAFII250 kinase activity. We further found that the Rb-related protein p107 can inhibit TAFII250 kinase activity, and this inhibition is likewise alleviated by cyclin D1. Cyclin D1 prevents the kinase-inhibitory effect of an Rb mutant unable to bind to D-type cyclins, indicating that it is acting through its association with TAFII250 and not with Rb. However, we found no evidence of TAFII250-binding competition between Rb and cyclin D1 in vitro. The adenovirus E1A protein, which also binds to both Rb and TAFII250, exhibited a suppressive effect on Rb-mediated kinase inhibition similar to that seen with cyclin D1. Our results suggest a novel means by which cyclin D1 may be able to independently regulate the activity of Rb.
Asunto(s)
Ciclina D1/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas , Proteína de Retinoblastoma/metabolismo , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animales , Unión Competitiva , Ciclina D1/genética , Ciclina D1/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Proteína de Retinoblastoma/antagonistas & inhibidores , Proteína de Retinoblastoma/genética , Proteína p107 Similar a la del Retinoblastoma , TATA BoxRESUMEN
Control of proliferation and differentiation by the retinoblastoma tumor suppressor protein (pRB) and related family members depends upon their interactions with key cellular substrates. Efforts to identify such cellular targets led to the isolation of a novel protein, EID-1 (for E1A-like inhibitor of differentiation 1). Here, we show that EID-1 is a potent inhibitor of differentiation and link this activity to its ability to inhibit p300 (and the highly related molecule, CREB-binding protein, or CBP) histone acetylation activity. EID-1 is rapidly degraded by the proteasome as cells exit the cell cycle. Ubiquitination of EID-1 requires an intact C-terminal region that is also necessary for stable binding to p300 and pRB, two proteins that bind to the ubiquitin ligase MDM2. A pRB variant that can bind to EID1, but not MDM2, stabilizes EID-1 in cells. Thus, EID-1 may act at a nodal point that couples cell cycle exit to the transcriptional activation of genes required for differentiation.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Ciclo Celular/fisiología , Proteínas de Saccharomyces cerevisiae , Acetiltransferasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteína de Unión a CREB , Proteínas de Ciclo Celular , Diferenciación Celular , Clonación Molecular , Regulación hacia Abajo , Histona Acetiltransferasas , Datos de Secuencia Molecular , Proteínas Nucleares/antagonistas & inhibidores , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Represoras , Proteína de Retinoblastoma/metabolismo , Transactivadores/antagonistas & inhibidores , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Ubiquitinas/metabolismoRESUMEN
PTEN acts as a tumor suppressor, at least in part, by antagonizing phosphoinositide 3-kinase (PI3K)/Akt signaling. Here we show that Forkhead transcription factors FKHRL1 and FKHR, substrates of the Akt kinase, are aberrantly localized to the cytoplasm and cannot activate transcription in PTEN-deficient cells. Restoration of PTEN function restores FKHR to the nucleus and restores transcriptional activation. Expression of a constitutively active form of FKHR that cannot be phosphorylated by Akt produces the same effect as reconstitution of PTEN on PTEN-deficient tumor cells. Specifically, activated FKHR induces apoptosis in cells that undergo PTEN-mediated cell death and induces G(1) arrest in cells that undergo PTEN-mediated cell cycle arrest. Furthermore, both PTEN and constitutively active FKHR induce p27(KIP1) protein but not p21. These data suggest that Forkhead transcription factors are critical effectors of PTEN-mediated tumor suppression.
Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/fisiología , Muerte Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Genes Supresores de Tumor , Monoéster Fosfórico Hidrolasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Transporte Biológico , Compartimento Celular , Núcleo Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Semivida , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfohidrolasa PTEN , Fosforilación , Transducción de Señal , Transcripción Genética , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Deregulation of cell cycle checkpoints is an almost universal abnormality in human cancers and is most often due to loss-of-function mutations of tumor suppressor genes such as Rb, p53, or p16(INK4a). In this study, we demonstrate that BCR/ABL inhibits the expression of a key cell cycle inhibitor, p27(Kip1), by signaling through a pathway involving phosphatidylinositol 3-kinase (PI3K). p27(Kip1) is a widely expressed inhibitor of cdk2, an essential cell cycle kinase regulating entry into S phase. We demonstrate that the decrease of p27(Kip1) is directly due to BCR/ABL in hematopoietic cells by two different approaches. First, induction of BCR/ABL by a tetracycline-regulated promoter is associated with a reversible down-regulation of p27(Kip1). Second, inhibition of BCR/ABL kinase activity with the Abl tyrosine kinase inhibitor STI571 rapidly increases p27(Kip1) levels. The PI3K inhibitor LY-294002 blocks the ability of BCR/ABL to induce p27(Kip1) down-regulation and inhibits BCR/ABL-induced entry into S phase. The serine/threonine kinase AKT/protein kinase B is a known downstream target of PI3K. Transient expression of an activated mutant of AKT was found to decrease expression of p27(Kip1), even when PI3K was inhibited by LY-294002. The mechanism of p27(Kip1) regulation is primarily related to protein stability, since inhibition of proteasome activity increased p27(Kip1) levels in BCR/ABL-transformed cells, whereas very little change in p27 transcription was found. Overall, these data are consistent with a model in which BCR/ABL suppresses p27(Kip1) protein levels through PI3K/AKT, leading to accelerated entry into S phase. This activity is likely to explain in part previous studies showing that activation of PI3K was required for optimum transformation of hematopoietic cells by BCR/ABL in vitro and in vivo.
Asunto(s)
Proteínas de Ciclo Celular , Regulación hacia Abajo , Proteínas de Fusión bcr-abl/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor , Animales , Antibacterianos/farmacología , Benzamidas , Ciclo Celular , Línea Celular , Separación Celular , Cromonas/farmacología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Genes abl/genética , Mesilato de Imatinib , Interleucina-3/farmacología , Ratones , Morfolinas/farmacología , Piperazinas/farmacología , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Pirimidinas/farmacología , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S/efectos de los fármacos , Transducción de Señal , Sirolimus/farmacología , Factores de Tiempo , TransfecciónRESUMEN
The PTEN gene is a tumor suppressor localized in the frequently altered chromosomal region 10q23. The tumor suppressor function of the PTEN protein (PTEN) has been linked to its ability to dephosphorylate the lipid second-messenger phosphatidylinositol 3,4, 5-trisphosphate and phosphatidylinositol 3,4-bisphosphate and, by doing so, to antagonize the phosphoinositide 3-kinase pathway. The PTEN protein consists of an amino-terminal phosphatase domain, a lipid binding C2 domain, and a 50-amino-acid C-terminal domain (the "tail") of unknown function. A number of studies have shown that the tail is dispensable for both phosphatase activity and blocking cell growth. Here, we show that the PTEN tail is necessary for maintaining protein stability and that it also acts to inhibit PTEN function. Thus, removing the tail results in a loss of stability but does not result in a loss of function because the resultant protein is more active. Furthermore, tail-dependent regulation of stability and activity is linked to the phosphorylation of three residues (S380, T382, and T383) within the tail. Therefore, the tail is likely to mediate the regulation of PTEN function through phosphorylation.
Asunto(s)
Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Supresoras de Tumor , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ácido Aspártico , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead , Fase G1/genética , Genes Supresores de Tumor , Humanos , Datos de Secuencia Molecular , Mutación , Fosfohidrolasa PTEN , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Eliminación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales CultivadasAsunto(s)
Genes Supresores de Tumor , Inhibidores de las Quinasa Fosfoinosítidos-3 , Monoéster Fosfórico Hidrolasas/genética , Proteínas Proto-Oncogénicas , Proteínas Supresoras de Tumor , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Activación Enzimática , Proteína-Tirosina Quinasas de Adhesión Focal , Mutación de Línea Germinal , Neoplasias/genética , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadAsunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas , Animales , Caspasas/metabolismo , Genes p53 , Humanos , FN-kappa B/efectos de los fármacos , FN-kappa B/fisiología , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-aktRESUMEN
The tumor suppressor gene PTEN/MMAC-1/TEP-1 (referred to hereafter as PTEN) maps to chromosome 10q23 and encodes a dual specificity phosphatase. The PTEN protein negatively regulates cell migration and cell survival and induces a G1 cell cycle block via negative regulation of the phosphatidylinositol 3'-kinase/protein kinase B/Akt signaling pathway. PTEN is frequently mutated or deleted in both prostate cancer cell lines and primary prostate cancers. A murine polyclonal antiserum was raised against a glutathione S-transferase fusion polypeptide of the COOH terninus of PTEN. Archival paraffin tissue sections from 109 cases of resected prostate cancer were immunostained with the antiserum, using DU145 and PC-3 cells as positive and negative controls, respectively. PTEN expression was seen in the secretory cells. Cases were considered positive when granular cytoplasmic staining was seen in all tumor cells, mixed when areas of both positive and negative tumor cell clones were seen, and negative when adjacent benign prostate tissue but not tumor tissue showed positive staining. Seventeen cases (15.6%) of prostate cancer were positive, 70 cases (64.2%) were mixed, and 22 cases (20.2%) were negative. Total absence of PTEN expression correlated with the Gleason score (P = 0.0081) and correlated more significantly with a Gleason score of 7 or higher (P = 0.0004) and with advanced pathological stage (American Joint Committee on Cancer stages T3b and T4; P = 0.0078). Thus, loss of PTEN protein is correlated with pathological markers of poor prognosis in prostate cancer.
Asunto(s)
Monoéster Fosfórico Hidrolasas/análisis , Neoplasias de la Próstata/metabolismo , Proteínas Supresoras de Tumor , Adulto , Anciano , Anciano de 80 o más Años , Genes Supresores de Tumor , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Fosfohidrolasa PTEN , Parafina , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/fisiología , Neoplasias de la Próstata/patología , Adhesión del Tejido , Células Tumorales CultivadasRESUMEN
PTEN/MMAC1 is a tumor suppressor gene located on chromosome 10q23. Inherited PTEN/MMAC1 mutations are associated with a cancer predisposition syndrome known as Cowden's disease. Somatic mutation of PTEN has been found in a number of malignancies, including glioblastoma, melanoma, and carcinoma of the prostate and endometrium. The protein product (PTEN) encodes a dual-specificity protein phosphatase and in addition can dephosphorylate certain lipid substrates. Herein, we show that PTEN protein induces a G1 block when reconstituted in PTEN-null cells. A PTEN mutant associated with Cowden's disease (PTEN;G129E) has protein phosphatase activity yet is defective in dephosphorylating inositol 1,3,4,5-tetrakisphosphate in vitro and fails to arrest cells in G1. These data suggest a link between induction of a cell-cycle block by PTEN and its ability to dephosphorylate, in vivo, phosphatidylinositol 3,4,5-trisphosphate. In keeping with this notion, PTEN can inhibit the phosphatidylinositol 3,4, 5-trisphosphate-dependent Akt kinase, a downstream target of phosphatidylinositol 3-kinase, and constitutively active, but not wild-type, Akt overrides a PTEN G1 arrest. Finally, tumor cells lacking PTEN contain high levels of activated Akt, suggesting that PTEN is necessary for the appropriate regulation of the phosphatidylinositol 3-kinase/Akt pathway.
Asunto(s)
Ciclo Celular/fisiología , Genes Supresores de Tumor , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor , Ciclo Celular/genética , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 10 , Fase G1 , Síndrome de Hamartoma Múltiple/genética , Humanos , Mutagénesis Sitio-Dirigida , Neoplasias/genética , Fosfohidrolasa PTEN , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteína de Retinoblastoma/química , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Quinasas Ciclina-Dependientes/química , Ciclinas/química , Cartilla de ADN/genética , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/genética , Especificidad por SustratoRESUMEN
The retinoblastoma tumor suppressor protein (pRB) can inhibit cell cycle progression and promote differentiation. pRB interacts with a variety of transcription factors, including members of the E2F and C-EBP protein families and MyoD, and can either repress or activate transcription depending on the promoter under study. These biological and biochemical activities of pRB have been mapped previously to a core domain, referred to as the pRB pocket. Using a panel of synthetic pRB pocket mutants, we found that the acute induction of a G1/S block by pRB is linked to its ability to both bind to E2F and to repress transcription. In contrast, these functions were not required for pRB to promote differentiation, which correlated with its ability to activate transcription in concert with fate-determining proteins such as MyoD. All tumor-derived pRB mutants tested to date failed to bind to E2F and did not repress transcription. Despite an inability to bind to E2F, pRB mutants associated with a low risk of retinoblastoma, unlike high-risk mutants, retained the ability to activate transcription and promote differentiation. Thus, the pRB pocket participates in dual tumor suppressor functions, one linked to cell cycle progression and the other to differentiation control, and these functions can be genetically and mechanistically dissociated.