RESUMEN
To assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm(-/-) mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1ß, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1ß or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy.
Asunto(s)
Mitocondrias , Telomerasa/antagonistas & inhibidores , Homeostasis del Telómero , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Genes cdc , Humanos , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Ratones , Mitocondrias/metabolismo , Invasividad Neoplásica/patología , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Receptores de Estrógenos/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-ß/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.
Asunto(s)
Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Telomerasa/metabolismo , Telómero/metabolismo , Animales , Neoplasias Óseas/secundario , Línea Celular Tumoral , Cruzamientos Genéticos , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Femenino , Inestabilidad Genómica , Humanos , Masculino , Ratones , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFß/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.
Asunto(s)
Progresión de la Enfermedad , Metástasis de la Neoplasia/patología , Neoplasias de la Próstata/patología , Proteína Smad4/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proliferación Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor/fisiología , Humanos , Neoplasias Pulmonares/secundario , Metástasis Linfática , Masculino , Ratones , Ratones Transgénicos , Modelos Biológicos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/genética , Osteopontina/genética , Osteopontina/metabolismo , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Penetrancia , Pronóstico , Próstata/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Proteína Smad4/deficiencia , Proteína Smad4/genética , Factor de Crecimiento Transformador betaRESUMEN
BACKGROUND: We previously identified a protein tumor signature of PTEN, SMAD4, SPP1, and CCND1 that, together with clinical features, was associated with lethal outcomes among prostate cancer patients. In the current study, we sought to validate the molecular model using time-dependent measures of AUC and predictive values for discriminating lethal from non-lethal prostate cancer. METHODS: Using data from the initial study, we fit survival models for men with prostate cancer who were participants in the Physicians' Health Study (PHS; n = 276). Based on these models, we generated prognostic risk scores in an independent population, the Health Professionals Follow-up Study (HPFS; n = 347) to evaluate external validity. In each cohort, men were followed prospectively from cancer diagnosis through 2011 for development of distant metastasis or cancer mortality. We measured protein tumor expression of PTEN, SMAD4, SPP1, and CCND1 on tissue microarrays. RESULTS: During a median of 11.9 and 14.3 years follow-up in the PHS and HPFS cohorts, 24 and 32 men (9%) developed lethal disease. When used as a prognostic factor in a new population, addition of the four markers to clinical variables did not improve discriminatory accuracy through 15 years of follow-up. CONCLUSIONS: Although the four markers have been identified as key biological mediators in metastatic progression, they do not provide independent, long-term prognostic information beyond clinical factors when measured at diagnosis. This finding may underscore the broad heterogeneity in aggressive prostate tumors and highlight the challenges that may result from overfitting in discovery-based research.
Asunto(s)
Ciclina D1/metabolismo , Osteopontina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/diagnóstico , Proteína Smad4/metabolismo , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Biomarcadores de Tumor/metabolismo , Progresión de la Enfermedad , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/patología , Pronóstico , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/mortalidadRESUMEN
Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53(-/-) Pten(-/-)) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53(-/-) Pten(-/-) TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.
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Neoplasias Encefálicas/patología , Diferenciación Celular , Glioma/patología , Células Madre Neoplásicas/patología , Neuronas/patología , Fosfohidrolasa PTEN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Neoplasias Encefálicas/genética , Proliferación Celular , Regulación de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Glioma/genética , Humanos , Inmunohistoquímica , Ratones , Células Madre Neoplásicas/metabolismo , Neuronas/metabolismo , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
mi-er1 (previously called er1) was first isolated from Xenopus laevis embryonic cells as a novel fibroblast growth factor-regulated immediate-early gene. Xmi-er1 was shown to encode a nuclear protein with an N-terminal acidic transcription activation domain. The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level and was shown to be upregulated in breast carcinoma cell lines and tumors. Alternative splicing at the 3' end of hmi-er1 produces two major isoforms, hMI-ER1alpha and hMI-ER1beta, which contain distinct C-terminal domains. In this study, we investigated the role of hMI-ER1alpha and hMI-ER1beta in the regulation of transcription. Using fusion proteins of hMI-ER1alpha or hMI-ER1beta tethered to the GAL4 DNA binding domain, we show that both isoforms, when recruited to the G5tkCAT minimal promoter, function to repress transcription. We demonstrate that this repressor activity is due to interaction and recruitment of a trichostatin A-sensitive histone deacetylase 1 (HDAC1). Furthermore, deletion analysis revealed that recruitment of HDAC1 to hMI-ER1alpha and hMI-ER1beta occurs through their common ELM2 domain. The ELM2 domain was first described in the Caenorhabditis elegans Egl-27 protein and is present in a number of SANT domain-containing transcription factors. This is the first report of a function for the ELM2 domain, highlighting its role in the regulation of transcription.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Unión al ADN , Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carcinoma/genética , Carcinoma/metabolismo , Secuencia Conservada , Inhibidores Enzimáticos/farmacología , Femenino , Proteínas del Helminto/química , Histona Desacetilasa 1 , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/genética , Humanos , Ácidos Hidroxámicos/farmacología , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Transcripción Genética , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/ß-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (ß-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of ß-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on ß-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on ß-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) ß-catenin were significantly more sensitive to ß-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active ß-catenin. Finally, significant therapeutic benefit of ß-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. ß-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of ß-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant ß-catenin signaling in the maintenance of oncogenic phenotype in HCC.
RESUMEN
UNLABELLED: The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo-YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP-TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5-CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer. SIGNIFICANCE: We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo-YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5-CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival.
Asunto(s)
Quimiocina CXCL5/genética , Células Mieloides/inmunología , Fosfohidrolasa PTEN/deficiencia , Neoplasias de la Próstata/inmunología , Proteína Smad4/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular Tumoral , Quimiocina CXCL5/metabolismo , Progresión de la Enfermedad , Vía de Señalización Hippo , Humanos , Masculino , Ratones , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 Pten(L/L) p53(L/L) LSL-mTERT(L/L) and thymic T-cell lymphoma model G4 Atm(-/-) mTERT(ER/ER). The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.
Asunto(s)
Alelos , Neoplasias/genética , Regeneración/genética , Telomerasa/genética , Animales , Células Cultivadas , Femenino , Técnicas de Sustitución del Gen/métodos , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/patología , Células-Madre Neurales/citología , Células-Madre Neurales/enzimología , Telómero/metabolismoRESUMEN
mi-er1 (previously called er1) is a fibroblast growth factor-inducible early response gene activated during mesoderm induction in Xenopus embryos and encoding a nuclear protein that functions as a transcriptional activator. The human orthologue of mi-er1 was shown to be upregulated in breast carcinoma cell lines and breast tumours when compared to normal breast cells. In this report, we investigate the structure of the human mi-er1 (hmi-er1) gene and characterize the alternatively spliced transcripts and protein isoforms. hmi-er1 is a single copy gene located at 1p31.2 and spanning 63 kb. It contains 17 exons and includes one skipped exon, a facultative intron and three polyadenylation signals to produce 12 transcripts encoding six distinct proteins. hmi-er1 transcripts were expressed at very low levels in most human adult tissues and the mRNA isoform pattern varied with the tissue. The 12 transcripts encode proteins containing a common internal sequence with variable N- and C-termini. Three distinct N- and two distinct C-termini were identified, giving rise to six protein isoforms. The two C-termini differ significantly in size and sequence and arise from alternate use of a facultative intron to produce hMI-ER1alpha and hMI-ER1beta. In all tissues except testis, transcripts encoding the beta isoform were predominant. hMI-ER1alpha lacks the predicted nuclear localization signal and transfection assays revealed that, unlike hMI-ER1beta, it is not a nuclear protein, but remains in the cytoplasm. Our results demonstrate that alternate use of a facultative intron regulates the subcellular localization of hMI-ER1 proteins and this may have important implications for hMI-ER1 function.
Asunto(s)
Empalme Alternativo , Proteínas Inmediatas-Precoces/genética , Proteínas Nucleares/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN , Femenino , Expresión Génica , Genes/genética , Humanos , Intrones/genética , Masculino , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción , Transcripción GenéticaRESUMEN
Sanguinarine, a benzophenanthrine alkaloid, is potentially antineoplastic through induction of cell death pathways. The development of multidrug resistance (MDR) is a major obstacle to the success of chemotherapeutic agents. The aim of this study was to investigate whether sanguinarine is effective against uterine cervical MDR and, if so, by which mechanism. The effects of treatment with sanguinarine on human papillomavirus (HPV) type 16-immortalized endocervical cells and their MDR counterpart cells were compared. Trypan blue exclusion assays and clonogenic survival assays demonstrated that MDR human cervical cells are as sensitive as their drug-sensitive parental cells to death induced by sanguinarine. Upon treatment of both types of cells with sanguinarine, two distinct concentration-dependent modes of cell death were observed. Treatment with 2.12 or 4.24 microM sanguinarine induced death in most cells that was characterized as apoptosis using the criteria of cell surface blebbing, as determined by light and scanning electron microscopy, and proteolytic activation of caspase-3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), as detected by Western blot analysis. However, 8.48 and 16.96 microM sanguinarine caused a second mode of cell death, oncosis, distinguished by cell surface blistering, and neither caspase-3 activation nor PARP cleavage. This study provides the first evidence that sanguinarine is effective against MDR in cervical cells via bimodal cell death, which displays alternative mechanisms involving different morphologies and caspase-3 activation status.
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Alcaloides/farmacología , Apoptosis , Cuello del Útero/citología , Sustancias Intercalantes/farmacología , Benzofenantridinas , Caspasa 3 , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Resistencia a Múltiples Medicamentos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , IsoquinolinasRESUMEN
mTORC1 is a validated therapeutic target for renal cell carcinoma (RCC). Here, analysis of Tsc1-deficient (mTORC1 hyperactivation) mice uncovered a FoxO-dependent negative feedback circuit constraining mTORC1-mediated renal tumorigenesis. We document robust FoxO activation in Tsc1-deficient benign polycystic kidneys and FoxO extinction on progression to murine renal tumors; murine renal tumor progression on genetic deletion of both Tsc1 and FoxOs; and downregulated FoxO expression in most human renal clear cell and papillary carcinomas, yet continued expression in less aggressive RCCs and benign renal tumor subtypes. Mechanistically, integrated analyses revealed that FoxO-mediated block operates via suppression of Myc through upregulation of the Myc antagonists, Mxi1-SRα and mir-145, establishing a FoxO-Mxi1-SRα/mir-145 axis as a major progression block in renal tumor development.
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Carcinoma de Células Renales/metabolismo , Factores de Transcripción Forkhead/fisiología , Neoplasias Renales/metabolismo , Proteínas/fisiología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Carcinoma de Células Renales/patología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , MicroARNs/genética , MicroARNs/metabolismo , MicroARNs/fisiología , Complejos Multiproteicos , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/fisiología , Transducción de Señal , Serina-Treonina Quinasas TOR , Activación Transcripcional , Proteína 1 del Complejo de la Esclerosis Tuberosa , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
The PI3K-AKT-FoxO pathway is integral to lifespan regulation in lower organisms and essential for the stability of long-lived cells in mammals. Here, we report the impact of combined FoxO1, 3, and 4 deficiencies on mammalian brain physiology with a particular emphasis on the study of the neural stem/progenitor cell (NSC) pool. We show that the FoxO family plays a prominent role in NSC proliferation and renewal. FoxO-deficient mice show initial increased brain size and proliferation of neural progenitor cells during early postnatal life, followed by precocious significant decline in the NSC pool and accompanying neurogenesis in adult brains. Mechanistically, integrated transcriptomic, promoter, and functional analyses of FoxO-deficient NSC cultures identified direct gene targets with known links to the regulation of human brain size and the control of cellular proliferation, differentiation, and oxidative defense. Thus, the FoxO family coordinately regulates diverse genes and pathways to govern key aspects of NSC homeostasis in the mammalian brain.
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Encéfalo/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre Multipotentes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Encéfalo/patología , Proteínas de Unión a Calmodulina , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Ratones , Ratones Noqueados , Células Madre Multipotentes/citología , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Tamaño de los Órganos/genética , Oxígeno/metabolismo , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Proteínas Wnt/genéticaRESUMEN
Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAF(V600E)--a mutation found in approximately 10% of human prostate tumors--was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAF(V600E) in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAF(V600E) is not required for its maintenance.
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Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Andrógenos , Animales , Biomarcadores de Tumor/metabolismo , Castración , Linaje de la Célula , Proliferación Celular , Células Epiteliales/enzimología , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Hiperplasia , Masculino , Ratones , Ratones Transgénicos , Invasividad Neoplásica , Fosfoproteínas/metabolismo , Próstata/enzimología , Próstata/patología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transactivadores/metabolismo , Transgenes , Urotelio/embriología , Urotelio/patologíaRESUMEN
Activated phosphoinositide 3-kinase (PI3K)-AKT signaling appears to be an obligate event in the development of cancer. The highly related members of the mammalian FoxO transcription factor family, FoxO1, FoxO3, and FoxO4, represent one of several effector arms of PI3K-AKT signaling, prompting genetic analysis of the role of FoxOs in the neoplastic phenotypes linked to PI3K-AKT activation. While germline or somatic deletion of up to five FoxO alleles produced remarkably modest neoplastic phenotypes, broad somatic deletion of all FoxOs engendered a progressive cancer-prone condition characterized by thymic lymphomas and hemangiomas, demonstrating that the mammalian FoxOs are indeed bona fide tumor suppressors. Transcriptome and promoter analyses of differentially affected endothelium identified direct FoxO targets and revealed that FoxO regulation of these targets in vivo is highly context-specific, even in the same cell type. Functional studies validated Sprouty2 and PBX1, among others, as FoxO-regulated mediators of endothelial cell morphogenesis and vascular homeostasis.
Asunto(s)
Linaje de la Célula/genética , Transformación Celular Neoplásica/genética , Células Endoteliales/metabolismo , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Supresoras de Tumor/genética , Animales , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Transformación Celular Neoplásica/metabolismo , Proteínas de Drosophila , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Hemangioma/genética , Hemangioma/metabolismo , Proteínas de Homeodominio/genética , Homeostasis/genética , Linfoma/genética , Linfoma/metabolismo , Ratones , Ratones Noqueados , Neovascularización Fisiológica/genética , Proteínas del Tejido Nervioso/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
To gain insight into the regulation of hmi-er1 expression, we cloned a human genomic DNA fragment containing one of the two hmi-er1 promoters and consisting of 1460 bp upstream of the translation initiation codon of hMI-ER1. Computer-assisted sequence analysis revealed that the hmi-er1 promoter region contains a CpG island but lacks an identifiable TATA element, initiator sequence and downstream promoter element. This genomic DNA was able to direct transcription of a luciferase reporter gene in a variety of human cell lines, and the minimal promoter was shown to be located within-68/+144 bp. Several putative Sp1 binding sites were identified, and we show that Sp1 can bind to the hmi-er1 minimal promoter and increase transcription, suggesting that the level of hmi-er1 expression may depend on the availability of Sp1 protein. Functional analysis revealed that hMI-ER1 represses Sp1-activated transcription from the minimal promoter by a histone deacetylase-independent mechanism. Chromatin immunoprecipitation analysis demonstrated that both Sp1 and hMI-ER1 are associated with the chromatin of the hmi-er1 promoter and that overexpression of hMI-ER1 in cell lines that allow Tet-On-inducible expression resulted in loss of detectable Sp1 from the endogenous hmi-er1 promoter. The mechanism by which this occurs does not involve binding of hMI-ER1 to cis-acting elements. Instead, we show that hMI-ER1 physically associates with Sp1 and that endogenous complexes containing the two proteins could be detected in vivo. Furthermore, hMI-ER1 specifically interferes with binding of Sp1 to the hmi-er1 minimal promoter as well as to an Sp1 consensus oligonucleotide. Deletion analysis revealed that this interaction occurs through a region containing the SANT domain of hMI-ER1. Together, these data reveal a functional role for the SANT domain in the action of co-repressor regulatory factors and suggest that the association of hMI-ER1 with Sp1 represents a novel mechanism for the negative regulation of Sp1 target promoters.
Asunto(s)
Proteínas Inmediatas-Precoces/química , Proteínas Nucleares/química , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , Cromatina/metabolismo , Clonación Molecular , Islas de CpG , ADN/genética , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Genes Reporteros , Glutatión Transferasa/metabolismo , Células HeLa , Histona Desacetilasas/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Factores de Transcripción , Transcripción Genética , TransfecciónRESUMEN
Telomerase, a ribonucleoprotein complex of hTERT, hTR, and TP1, has been reported to be associated with carcinogenesis and multidrug resistance (MDR). This study used our in vitro human cervical multistep carcinogenesis/MDR model system in which normal human ectocervical and endocervical (HEN) cells were immortalized by HPV18 or 16, respectively, and subsequently transformed. The first evidence was found that immortalization and telomerase activation were correlated with increased expression specifically of two of the hTERT alternatively spliced mRNAs, one encoding wild-type protein containing the full-length functional reverse transcriptase (RT) region and one encoding a defective RT protein. Expression of neither hTERT mRNA containing full-length functional or defective RT motif was affected by transformation/MDR. All-trans-retinoic acid (ATRA) treatment of HPV-immortalized HEN-16-2 cells and transformed/MDR HEN-16-2/CDDP cells inhibited telomerase activity and downregulated expression of hTERT mRNAs containing full-length functional and a defective RT motif, but there were no changes in hTR and TP1 expression. Moreover, ATRA inhibited cell growth rate of HEN-16-2 and HEN-16-2/CDDP cells equally. These results provided the first evidence that ATRA equally in both immortalized and transformed/MDR cell lines inhibits telomerase activity and downregulates expression, but not splicing, of hTERT, and this is correlated with cell growth rate inhibition; the potential is implicated for applying ATRA to hTERT-targeted treatment of cervical cell carcinogenesis/MDR.