RESUMEN
BACKGROUND: Human cancer cell line profiling and drug sensitivity studies provide valuable information about the therapeutic potential of drugs and their possible mechanisms of action. The goal of those studies is to translate the findings from in vitro studies of cancer cell lines into in vivo therapeutic relevance and, eventually, patients' care. Tremendous progress has been made. RESULTS: In this work, we built predictive models for 453 drugs using data on gene expression and drug sensitivity (IC50) from cancer cell lines. We identified many known drug-gene interactions and uncovered several potentially novel drug-gene associations. Importantly, we further applied these predictive models to ~ 17,000 bulk RNA-seq samples from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database to predict drug sensitivity for both normal and tumor tissues. We created a web site for users to visualize and download our predicted data ( https://manticore.niehs.nih.gov/cancerRxTissue ). Using trametinib as an example, we showed that our approach can faithfully recapitulate the known tumor specificity of the drug. CONCLUSIONS: We demonstrated that our approach can predict drugs that 1) are tumor-type specific; 2) elicit higher sensitivity from tumor compared to corresponding normal tissue; 3) elicit differential sensitivity across breast cancer subtypes. If validated, our prediction could have relevance for preclinical drug testing and in phase I clinical design.
Asunto(s)
Neoplasias de la Mama , Preparaciones Farmacéuticas , Biomarcadores , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Expresión Génica , Perfilación de la Expresión Génica , HumanosRESUMEN
Quantifying cell-type proportions and their corresponding gene expression profiles in tissue samples would enhance understanding of the contributions of individual cell types to the physiological states of the tissue. Current approaches that address tissue heterogeneity have drawbacks. Experimental techniques, such as fluorescence-activated cell sorting, and single cell RNA sequencing are expensive. Computational approaches that use expression data from heterogeneous samples are promising, but most of the current methods estimate either cell-type proportions or cell-type-specific expression profiles by requiring the other as input. Although such partial deconvolution methods have been successfully applied to tumor samples, the additional input required may be unavailable. We introduce a novel complete deconvolution method, CDSeq, that uses only RNA-Seq data from bulk tissue samples to simultaneously estimate both cell-type proportions and cell-type-specific expression profiles. Using several synthetic and real experimental datasets with known cell-type composition and cell-type-specific expression profiles, we compared CDSeq's complete deconvolution performance with seven other established deconvolution methods. Complete deconvolution using CDSeq represents a substantial technical advance over partial deconvolution approaches and will be useful for studying cell mixtures in tissue samples. CDSeq is available at GitHub repository (MATLAB and Octave code): https://github.com/kkang7/CDSeq.
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Perfilación de la Expresión Génica/estadística & datos numéricos , Análisis de Secuencia de ARN/estadística & datos numéricos , Aprendizaje Automático no Supervisado , Línea Celular , Biología Computacional/métodos , Simulación por Computador , Bases de Datos de Ácidos Nucleicos/estadística & datos numéricos , Humanos , Leucocitos/clasificación , Leucocitos/metabolismo , Reconocimiento de Normas Patrones Automatizadas , TranscriptomaRESUMEN
Myocardin is known as an important transcriptional regulator in smooth and cardiac muscle development. Here we found that myocardin is frequently repressed during human malignant transformation, contributing to a differentiation defect. We demonstrate that myocardin is a transcriptional target of TGFbeta required for TGFbeta-mediated differentiation of human fibroblasts. Serum deprivation, intact contact inhibition response, and the p16ink4a/Rb pathway contribute to myocardin induction and differentiation. Restoration of myocardin expression in sarcoma cells results in differentiation and inhibition of malignant growth, whereas inactivation of myocardin in normal fibroblasts increases their proliferative potential. Myocardin expression is reduced in multiple types of human tumors. Collectively, our results demonstrate that myocardin is an important suppressive modifier of the malignant transformation process.
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Diferenciación Celular , Transformación Celular Neoplásica , Inhibidor p16 de la Quinasa Dependiente de Ciclina/antagonistas & inhibidores , Fibroblastos/citología , Proteínas Nucleares/antagonistas & inhibidores , Transactivadores/antagonistas & inhibidores , Western Blotting , Adhesión Celular , Proliferación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilación de ADN , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Humanos , Pulmón/embriología , Mesodermo/citología , Mesodermo/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plásmidos , Regiones Promotoras Genéticas , ARN Interferente Pequeño/farmacología , Transactivadores/genética , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Previous work has identified distinct functions for E2F proteins during a cellular proliferative response including a role for E2F1-3 in the activation of transcription at G1/S and a role for E2F4-8 in repressing the same group of E2F1-3 target genes as cells progress through S phase. We now find that E2F7 and E2F8, which are induced by E2F1-3 at G1/S, can form a heterodimer with E2F1 through interactions involving the DNA-binding domains of the two proteins. In vitro DNA interaction assays demonstrate the formation of an E2F1-E2F7 complex, as well as an E2F7-E2F7 complex on adjacent E2F-binding sites. We also show that E2F7 recruits the co-repressor C-terminal-binding protein (CtBP) and that CtBP2 is essential for E2F7 to repress E2F1 transcription. Taken together, these findings suggest a mechanism for the repression of transcription by E2F7.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F7/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética/fisiología , Oxidorreductasas de Alcohol/genética , Línea Celular Tumoral , Proteínas Co-Represoras , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F7/genética , Fase G1/fisiología , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Proteínas del Tejido Nervioso/genética , Multimerización de Proteína/fisiología , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Fase S/fisiologíaRESUMEN
DNASE1L3, an enzyme highly expressed in DCs, is functionally important for regulating autoimmune responses to self-DNA and chromatin. Deficiency of DNASE1L3 leads to development of autoimmune diseases in both humans and mice. However, despite the well-established causal relationship between DNASE1L3 and immunity, little is known about the involvement of DNASE1L3 in regulation of antitumor immunity, the foundation of modern antitumor immunotherapy. In this study, we identify DNASE1L3 as a potentially new regulator of antitumor immunity and a tumor suppressor in colon cancer. In humans, DNASE1L3 is downregulated in tumor-infiltrating DCs, and this downregulation is associated with poor patient prognosis and reduced tumor immune cell infiltration in many cancer types. In mice, Dnase1l3 deficiency in the tumor microenvironment enhances tumor formation and growth in several colon cancer models. Notably, the increased tumor formation and growth in Dnase1l3-deficient mice are associated with impaired antitumor immunity, as evidenced by a substantial reduction of cytotoxic T cells and a unique subset of DCs. Consistently, Dnase1l3-deficient DCs directly modulate cytotoxic T cells in vitro. To our knowledge, our study unveils a previously unknown link between DNASE1L3 and antitumor immunity and further suggests that restoration of DNASE1L3 activity may represent a potential therapeutic approach for anticancer therapy.
Asunto(s)
Neoplasias del Colon , Humanos , Ratones , Animales , Neoplasias del Colon/metabolismo , Cromatina/metabolismo , Inmunoterapia , Linfocitos T Citotóxicos , Microambiente Tumoral , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismoRESUMEN
Restriction of methionine (MR), a sulfur-containing essential amino acid, has been reported to repress cancer growth and improve therapeutic responses in several preclinical settings. However, how MR impacts cancer progression in the context of the intact immune system is unknown. Here we report that while inhibiting cancer growth in immunocompromised mice, MR reduces T cell abundance, exacerbates tumour growth and impairs tumour response to immunotherapy in immunocompetent male and female mice. Mechanistically, MR reduces microbial production of hydrogen sulfide, which is critical for immune cell survival/activation. Dietary supplementation of a hydrogen sulfide donor or a precursor, or methionine, stimulates antitumour immunity and suppresses tumour progression. Our findings reveal an unexpected negative interaction between MR, sulfur deficiency and antitumour immunity and further uncover a vital role of gut microbiota in mediating this interaction. Our study suggests that any possible anticancer benefits of MR require careful consideration of both the microbiota and the immune system.
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Microbioma Gastrointestinal , Sulfuro de Hidrógeno , Neoplasias , Masculino , Ratones , Femenino , Animales , Metionina/metabolismo , Sulfuro de Hidrógeno/metabolismo , Racemetionina , AzufreRESUMEN
Methionine restriction, a dietary regimen that protects against metabolic diseases and aging, represses cancer growth and improves cancer therapy. However, the response of different cancer cells to this nutritional manipulation is highly variable, and the molecular determinants of this heterogeneity remain poorly understood. Here we report that hepatocyte nuclear factor 4α (HNF4α) dictates the sensitivity of liver cancer to methionine restriction. We show that hepatic sulfur amino acid (SAA) metabolism is under transcriptional control of HNF4α. Knocking down HNF4α or SAA enzymes in HNF4α-positive epithelial liver cancer lines impairs SAA metabolism, increases resistance to methionine restriction or sorafenib, promotes epithelial-mesenchymal transition, and induces cell migration. Conversely, genetic or metabolic restoration of the transsulfuration pathway in SAA metabolism significantly alleviates the outcomes induced by HNF4α deficiency in liver cancer cells. Our study identifies HNF4α as a regulator of hepatic SAA metabolism that regulates the sensitivity of liver cancer to methionine restriction.
Asunto(s)
Factor Nuclear 4 del Hepatocito/metabolismo , Neoplasias Hepáticas/metabolismo , Metionina/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Cisteína/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor Nuclear 4 del Hepatocito/genética , Hígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Mesodermo/efectos de los fármacos , Mesodermo/patología , Redes y Vías Metabólicas/efectos de los fármacos , Metaboloma/efectos de los fármacos , Ratones , Sorafenib/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.
Asunto(s)
Amidas/metabolismo , Vías Biosintéticas , Mamíferos/microbiología , Mycoplasma/fisiología , NAD/metabolismo , Administración Oral , Animales , Línea Celular Tumoral , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Metabolismo Energético , Femenino , Microbioma Gastrointestinal , Humanos , Masculino , Metaboloma , Ratones Endogámicos C57BL , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Nicotinamidasa/metabolismo , Mononucleótido de Nicotinamida/administración & dosificación , Mononucleótido de Nicotinamida/química , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Compuestos de Piridinio/metabolismoRESUMEN
Due to the poor prognosis of advanced metastatic melanoma, it is crucial to find early biomarkers that help identify which melanomas will metastasize. By comparing the gene expression data from primary and cutaneous melanoma samples from The Cancer Genome Atlas (TCGA), we identified GPC6 among a set of genes whose expression levels can distinguish between primary melanoma and regional cutaneous/subcutaneous metastases. Glypicans are thought to play a role in tumor growth by regulating the signaling pathways of Wnt, Hedgehogs, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). We showed that GPC6 expression was up-regulated in a melanoma cell line compared to normal melanocytes and in metastatic melanoma compared to primary melanoma. Furthermore, GPC6 expression was positively correlated with genes largely involved in cell adhesion and migration in both melanoma samples and in RNA-seq samples from other TCGA tumors. Our results suggest that GPC6 may play a role in tumor metastatic progression. In TCGA melanoma samples, we also showed that GPC6 expression was negatively correlated with miR-509-3p, which has previously been shown to function as a tumor suppressor in various cancer cell lines. We overexpressed miR-509-3p in A375 melanoma cells and showed that GPC6 expression was significantly suppressed. This result suggested that GPC6 was a putative target of miR-509-3p in melanoma. Together, our findings identified GPC6 as an early biomarker for melanoma metastatic progression, one that can be regulated by miR-509-3p.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Regulación Neoplásica de la Expresión Génica , Glipicanos/biosíntesis , Melanoma/metabolismo , Proteínas de Neoplasias/biosíntesis , Neoplasias Cutáneas/metabolismo , Regulación hacia Arriba , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Femenino , Glipicanos/genética , Humanos , Masculino , Melanoma/genética , Melanoma/patología , MicroARNs/biosíntesis , MicroARNs/genética , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Melanoma Cutáneo MalignoRESUMEN
Prostate cancer is the most commonly diagnosed type of cancer in men, and there is no available cure for patients with advanced disease. In vitro model systems are urgently required to permit the study of human prostate cell differentiation and malignant transformation. Unfortunately, human prostate cells are particularly difficult to convert into continuously growing cultures. We report here the successful immortalization without viral oncogenes of prostate epithelial cells and, for the first time, prostate stromal cells. These cells exhibit a significant pattern of authentic prostate-specific features. In particular, the epithelial cell culture is able to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells. The stromal cells have typical characteristics of prostate smooth muscle cells. These immortalized cultures may serve as a unique experimental platform to permit several research directions, including the study of cell-cell interactions in an authentic prostate microenvironment, prostate cell differentiation, and most significantly, the complex multistep process leading to prostate cell transformation.
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Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/fisiología , Próstata/citología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Telomerasa/fisiología , Anciano , Diferenciación Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Humanos , Masculino , Próstata/fisiología , Neoplasias de la Próstata/genética , Células del Estroma/citología , Células del Estroma/metabolismo , Células del Estroma/fisiología , Telomerasa/biosíntesis , Telomerasa/genética , Transfección , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Mutations in p53 are ubiquitous in human tumors. Some p53 mutations not only result in loss of wild-type (WT) activity but also grant additional functions, termed "gain of function." In this study, we explore how the status of p53 affects the immediate response gene activating transcription factor 3 (ATF3) in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-protein kinase C (PKC) pathway. We show that high doses of TPA induce ATF3 in a WT p53-independent manner correlating with PKCs depletion and cell death. We show that cells harboring mutant p53 have attenuated ATF3 induction and are less sensitive to TPA-induced death compared with their p53-null counterparts. Mutagenesis analysis of the ATF3 promoter identified the regulatory motifs cyclic AMP-responsive element binding protein/ATF and MEF2 as being responsible for the TPA-induced activation of ATF3. Moreover, we show that mutant p53 attenuates ATF3 expression by two complementary mechanisms. It interacts with the ATF3 promoter and influences its activity via the MEF2 site, and additionally, it attenuates transcriptional expression of the ATF3 activator MEF2D. These data provide important insights into the molecular mechanisms that underlie mutant p53 gain of function.
Asunto(s)
Factor de Transcripción Activador 3/biosíntesis , Mutación , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Factor de Transcripción Activador 3/antagonistas & inhibidores , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Secuencia de Bases , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción MEF2 , Datos de Secuencia Molecular , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Regiones Promotoras Genéticas , Acetato de Tetradecanoilforbol/farmacología , Transfección , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The difficulty to dissect a complex phenotype of established malignant cells to several critical transcriptional programs greatly impedes our understanding of the malignant transformation. The genetic elements required to transform some primary human cells to a tumorigenic state were described in several recent studies. We took the advantage of the global genomic profiling approach and tried to go one step further in the dissection of the transformation network. We sought to identify the genetic signatures and key target genes, which underlie the genetic alterations in p53, Ras, INK4A locus, and telomerase, introduced in a stepwise manner into primary human fibroblasts. Here, we show that these are the minimally required genetic alterations for sarcomagenesis in vivo. A genome-wide expression profiling identified distinct genetic signatures corresponding to the genetic alterations listed above. Most importantly, unique transformation hallmarks, such as differentiation block, aberrant mitotic progression, increased angiogenesis, and invasiveness, were identified and coupled with genetic signatures assigned for the genetic alterations in the p53, INK4A locus, and H-Ras, respectively. Furthermore, a transcriptional program that defines the cellular response to p53 inactivation was an excellent predictor of metastasis development and bad prognosis in breast cancer patients. Deciphering these transformation fingerprints, which are affected by the most common oncogenic mutations, provides considerable insight into regulatory circuits controlling malignant transformation and will hopefully open new avenues for rational therapeutic decisions.
Asunto(s)
Transformación Celular Neoplásica/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Genes p53/genética , Genes ras/genética , Transcripción Genética/genética , Diferenciación Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Proteínas de Unión al ADN , Fibroblastos/citología , Fibroblastos/fisiología , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Pulmón/citología , Pulmón/fisiología , Telomerasa/genéticaRESUMEN
The Rb/E2F network has a critical role in regulating cell cycle progression and cell fate decisions. It is dysfunctional in virtually all human cancers, because of genetic lesions that cause overexpression of activators, inactivation of repressors, or both. Paradoxically, the downstream target of this network, E2F1, is rarely strongly overexpressed in cancer. E2F1 can induce both proliferation and apoptosis but the factors governing these critical cell fate decisions remain unclear. Previous studies have focused on qualitative mechanisms such as differential cofactors, posttranslational modification or state of other signaling pathways as modifiers of the cell fate decisions downstream of E2F1 activation. In contrast, the importance of the expression levels of E2F1 itself in dictating the downstream phenotypes has not been rigorously studied, partly due to the limited resolution of traditional population-level measurements. Here, through single-cell quantitative analysis, we demonstrate that E2F1 expression levels have a critical role in determining the fate of individual cells. Low levels of exogenous E2F1 promote proliferation, moderate levels induce G1, G2 and mitotic cell cycle arrest, and very high levels promote apoptosis. These multiple anti-proliferative mechanisms result in a strong selection pressure leading to rapid elimination of E2F1-overexpressing cells from the population. RNA-sequencing and RT-PCR revealed that low levels of E2F1 are sufficient to induce numerous cell cycle-promoting genes, intermediate levels induce growth arrest genes (i.e., p18, p19 and p27), whereas higher levels are necessary to induce key apoptotic E2F1 targets APAF1, PUMA, HRK and BIM. Finally, treatment of a lung cancer cell line with a proteasome inhibitor, MLN2238, resulted in an E2F1-dependent mitotic arrest and apoptosis, confirming the role of endogenous E2F1 levels in these phenotypes. The strong anti-proliferative activity of moderately overexpressed E2F1 in multiple cancer types suggests that targeting E2F1 for upregulation may represent an attractive therapeutic strategy in cancer.
Asunto(s)
Apoptosis , Factor de Transcripción E2F1/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Factor Apoptótico 1 Activador de Proteasas/química , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Proteína 11 Similar a Bcl2/química , Proteína 11 Similar a Bcl2/metabolismo , Compuestos de Boro/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Factor de Transcripción E2F1/genética , Glicina/análogos & derivados , Glicina/farmacología , Células HCT116 , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Tamoxifeno/toxicidad , Imagen de Lapso de TiempoRESUMEN
Deciphering regulatory events that drive malignant transformation represents a major challenge for systems biology. Here, we analyzed genome-wide transcription profiling of an in vitro cancerous transformation process. We focused on a cluster of genes whose expression levels increased as a function of p53 and p16(INK4A) tumor suppressors inactivation. This cluster predominantly consists of cell cycle genes and constitutes a signature of a diversity of cancers. By linking expression profiles of the genes in the cluster with the dynamic behavior of p53 and p16(INK4A), we identified a promoter architecture that integrates signals from the two tumor suppressive channels and that maps their activity onto distinct levels of expression of the cell cycle genes, which, in turn, correspond to different cellular proliferation rates. Taking components of the mitotic spindle as an example, we experimentally verified our predictions that p53-mediated transcriptional repression of several of these novel targets is dependent on the activities of p21, NFY, and E2F. Our study demonstrates how a well-controlled transformation process allows linking between gene expression, promoter architecture, and activity of upstream signaling molecules.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Transformación Celular Neoplásica/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Perfilación de la Expresión Génica , Genes Supresores de Tumor , Genes cdc , Regiones Promotoras Genéticas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Proteínas de Ciclo Celular/fisiología , División Celular , Línea Celular Transformada/metabolismo , Línea Celular Transformada/trasplante , Biología Computacional , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Genes p16 , Genes p53 , Humanos , Ratones , Ratones Desnudos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Huso Acromático/metabolismo , Telomerasa/genética , Telomerasa/fisiología , Transcripción Genética , Trasplante HeterólogoRESUMEN
Hypoxic stress is one of the major selective pressures in the microenvironment of solid tumors, and overcoming this restriction is essential for tumor progression. One of the key factors driving the cellular response to lack of oxygen is hypoxia inducible factor (HIF), a key transcriptional factor. The level of the alpha subunit of HIF-1 is regulated by rapid degradation that is controlled by a family of prolyl hydroxylases (PHDs/EGLNs), the activity of which depends on oxygen availability. Our study shows that ectopic expression of mPHD1 suppressed accumulation of HIF-1alpha and secretion of Vascular Endothelial Growth Factor after treatment of cells with a hypoxia-mimetic drug. Furthermore, when colon carcinoma cells expressing mPHD1 were injected into nude mice, tumor growth was inhibited, and the inhibition of tumor growth was correlated with increased necrosis and a striking decrease in microvessel density. These data demonstrate that inhibition of hypoxia-induced activation of HIF-1alpha through activation of HIF-hydroxylase can provide a novel therapeutic strategy for inhibition of tumor growth and neovascularization and support the development of gene transfer approaches based on the activation of HIF-prolyl hydroxylases.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Neoplasias Experimentales/patología , Procolágeno-Prolina Dioxigenasa/fisiología , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/antagonistas & inhibidores , Animales , División Celular/fisiología , Hipoxia de la Célula , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Deferoxamina/farmacología , Células HCT116 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/metabolismo , Procolágeno-Prolina Dioxigenasa/biosíntesis , Procolágeno-Prolina Dioxigenasa/genética , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Trasplante HeterólogoRESUMEN
Telomere shortening in primary human fibroblasts results in replicative senescence, which can be overcome by telomerase (hTERT) overexpression. However, because immortalization is one of the hallmarks of malignant transformation, careful analysis of hTERT-immortalized cells is of crucial importance for understanding both processes. To this end, we infected WI-38 fibroblasts with a retrovirus carrying the hTERT cDNA and analyzed their proliferative behavior during 600 days [ approximately 500 population doublings (PDLs)] of continuous culture. Growth of three independent mass cultures was uniform for approximately 150 PDLs after telomerase infection, followed by a progressive acceleration of growth in two of three cultures. Expression of p16(INK4A) was significantly elevated in the immortalized cells but gradually disappeared during the accelerated growth phase. This alteration correlated with loss of the contact inhibition response and conferred the cells with sensitivity to H-Ras-induced transformation. In contrast, the p53- and pRb-mediated checkpoints such as the DNA damage response, chromosomal stability and entry into quiescence remained intact, irrespective of INK4A locus expression. Importantly, detailed examination of one of the WI-38/hTERT cultures during the accelerated growth phase revealed overexpression of the c-myc and Bmi-1 oncogenes, as well as loss of p14(ARF) expression. Collectively, our results indicate that although hTERT-immortalized cells behave similarly to primary cells during the first 150 PDLs, long-term growth in culture may favor the appearance of clones carrying potentially malignant alterations.
Asunto(s)
Transformación Celular Neoplásica/patología , Lesiones Precancerosas/enzimología , Telomerasa/metabolismo , División Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Inhibición de Contacto , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Unión al ADN , Fibroblastos/citología , Fibroblastos/enzimología , Silenciador del Gen , Genes p53/fisiología , Genes ras/fisiología , Humanos , Lesiones Precancerosas/patología , Proteína de Retinoblastoma/genética , Retroviridae/enzimología , Retroviridae/genética , Telomerasa/biosíntesis , Telomerasa/genética , Factores de Tiempo , Transducción Genética , Proteína p14ARF Supresora de Tumor/biosíntesis , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
Replicative senescence is an irreversible cell cycle arrest that limits the proliferation of damaged cells and may be an important tumor suppression mechanism in vivo. This process is regulated at critical steps by the tumor suppressor p53. To identify genes that may regulate the senescence process, we performed cDNA microarray analysis of gene expression in senescent, young proliferating, and hTERT-immortalized primary human fibroblasts. The histone methyltransferase (HMTase), EZH2, was specifically downregulated in senescent cells. Activated p53 suppressed EZH2 gene expression through repression of the EZH2 gene promoter. This activity of p53 requires intact p53 transactivation and DNA binding domains. Furthermore, the repression of EZH2 promoter by p53 is dependent on p53 transcriptional target p21(Waf1) inactivating RB/E2F pathways. In addition, the knockdown of EZH2 expression retards cell proliferation and induces G2/M arrest. We suggest that the p53-dependent suppression of EZH2 expression is a novel pathway that contributes to p53-mediated G2/M arrest. EZH2 associated complex possesses HMTase activity and is involved in epigenetic regulation. Activated p53 suppresses EZH2 expression, suggesting a further role for p53 in epigenetic regulation and in the maintenance of genetic stability. Suppression of EZH2 expression in tumors by p53 may lead to novel approaches to control cancer progression.
Asunto(s)
Proteínas/genética , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , División Celular/efectos de los fármacos , División Celular/genética , Senescencia Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteínas de Unión al ADN , Regulación hacia Abajo , Doxorrubicina/farmacología , Proteína Potenciadora del Homólogo Zeste 2 , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fase G2/genética , Regulación de la Expresión Génica , Genes Supresores de Tumor , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Mitosis/genética , Datos de Secuencia Molecular , Complejo Represivo Polycomb 2 , Regiones Promotoras Genéticas , Proteína Metiltransferasas , Proteínas/metabolismo , Interferencia de ARN , Transducción de Señal , Telomerasa/metabolismo , Factores de Transcripción , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.
Asunto(s)
Apoptosis , Factor de Unión a CCAAT/genética , Factor de Transcripción E2F1/metabolismo , Transcripción Genética , Apoptosis/genética , Factor de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Sarcoma/genética , Activación Transcripcional/genética , Regulación hacia Arriba/genéticaRESUMEN
The p53 tumor suppressor regulates transcription of genes associated with diverse cellular functions including apoptosis, growth arrest, DNA repair and differentiation. Recently, we established that p53 can modulate expression of Toll-like receptor (TLR) innate immunity genes but the degree of cross-talk between p53 and TLR pathways remained unclear. Here, using gene expression profiling we characterize the global effect of p53 on the TLR5-mediated transcription in MCF7 cells. We found that combined activation of p53 and TLR5 pathways synergistically increases expression of over 200 genes, mostly associated with immunity and inflammation. The synergy was observed in several human cancer cells and primary lymphocytes. The p53-dependent amplification of transcriptional response to TLR5 activation required expression of NFκB subunit p65 and was mediated by several molecular mechanisms including increased phosphorylation of p38 MAP kinase, PI3K and STAT3 signaling. Additionally, p53 induction increased cytokine expression in response to TNFα, another activator of NFκB and MAP kinase pathways, suggesting a broad interaction between p53 and these signaling pathways. The expression of many synergistically induced genes is elevated in breast cancer patients responsive to chemotherapy. We suggest that p53's capacity to enhance immune response could be exploited to increase antitumor immunity and to improve cancer treatment.