RESUMEN
Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.
Asunto(s)
Adenosina Desaminasa , Primates/genética , Edición de ARN/genética , Proteínas de Unión al ARN , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Femenino , Genotipo , Células HEK293 , Humanos , Masculino , Ratones , Músculos/metabolismo , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Proteolisis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Análisis Espacio-Temporal , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
BACKGROUND: The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. SCOPE OF REVIEW: p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. MAJOR CONCLUSIONS: The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. GENERAL SIGNIFICANCE: The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets.
Asunto(s)
Apoptosis , Proliferación Celular , Isomerasa de Peptidilprolil/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/genética , Fosforilación , Estabilidad Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Ectrodactyly, or Split-Hand/Foot Malformation (SHFM), is a congenital condition characterized by the loss of central rays of hands and feet. The p63 and the DLX5;DLX6 transcription factors, expressed in the embryonic limb buds and ectoderm, are disease genes for these conditions. Mutations of p63 also cause the ectodermal dysplasia-ectrodactyly-cleft lip/palate (EEC) syndrome, comprising SHFM. Ectrodactyly is linked to defects of the apical ectodermal ridge (AER) of the developing limb buds. FGF8 is the key signaling molecule in this process, able to direct proximo-distal growth and patterning of the skeletal primordial of the limbs. In the limb buds of both p63 and Dlx5;Dlx6 murine models of SHFM, the AER is poorly stratified and FGF8 expression is severely reduced. We show here that the FGF8 locus is a downstream target of DLX5 and that FGF8 counteracts Pin1-ΔNp63α interaction. In vivo, lack of Pin1 leads to accumulation of the p63 protein in the embryonic limbs and ectoderm. We show also that ΔNp63α protein stability is negatively regulated by the interaction with the prolyl-isomerase Pin1, via proteasome-mediated degradation; p63 mutant proteins associated with SHFM or EEC syndromes are resistant to Pin1 action. Thus, DLX5, p63, Pin1 and FGF8 participate to the same time- and location-restricted regulatory loop essential for AER stratification, hence for normal patterning and skeletal morphogenesis of the limb buds. These results shed new light on the molecular mechanisms at the basis of the SHFM and EEC limb malformations.
Asunto(s)
Ectodermo/embriología , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/metabolismo , Deformidades Congénitas de las Extremidades/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Animales , Tipificación del Cuerpo , Línea Celular , Modelos Animales de Enfermedad , Ectodermo/metabolismo , Técnicas de Inactivación de Genes , Proteínas de Homeodominio/genética , Humanos , Esbozos de los Miembros/embriología , Deformidades Congénitas de las Extremidades/patología , Ratones , Peptidilprolil Isomerasa de Interacción con NIMA , Fosfoproteínas/genética , Estabilidad Proteica , Transactivadores/genéticaRESUMEN
AIM: Diabetes is a major driver of cardiovascular disease, but the underlying mechanisms remain elusive. Prolyl-isomerase Pin1 recognizes specific peptide bonds and modulates function of proteins altering cellular homoeostasis. The present study investigates Pin1 role in diabetes-induced vascular disease. METHODS AND RESULTS: In human aortic endothelial cells (HAECs) exposed to high glucose, up-regulation of Pin1-induced mitochondrial translocation of pro-oxidant adaptor p66(Shc) and subsequent organelle disruption. In this setting, Pin1 recognizes Ser-116 inhibitory phosphorylation of endothelial nitric oxide synthase (eNOS) leading to eNOS-caveolin-1 interaction and reduced NO availability. Pin1 also mediates hyperglycaemia-induced nuclear translocation of NF-κB p65, triggering VCAM-1, ICAM-1, and MCP-1 expression. Indeed, gene silencing of Pin1 in HAECs suppressed p66(Shc)-dependent ROS production, restored NO release and blunted NF-kB p65 nuclear translocation. Consistently, diabetic Pin1(-/-) mice were protected against mitochondrial oxidative stress, endothelial dysfunction, and vascular inflammation. Increased expression and activity of Pin1 were also found in peripheral blood monocytes isolated from diabetic patients when compared with age-matched healthy controls. Interestingly, enough, Pin1 up-regulation was associated with impaired flow-mediated dilation, increased urinary 8-iso-prostaglandin F2α and plasma levels of adhesion molecules. CONCLUSIONS: Pin1 drives diabetic vascular disease by causing mitochondrial oxidative stress, eNOS dysregulation as well as NF-kB-induced inflammation. These findings provide molecular insights for novel mechanism-based therapeutic strategies in patients with diabetes.
Asunto(s)
Angiopatías Diabéticas/prevención & control , Enfermedades Mitocondriales/prevención & control , Estrés Oxidativo/fisiología , Isomerasa de Peptidilprolil/fisiología , Análisis de Varianza , Animales , Aorta/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CCL2/metabolismo , Citocromos c/biosíntesis , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Técnicas de Silenciamiento del Gen , Glucosa/farmacología , Humanos , Hiperglucemia/fisiopatología , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Regulación hacia Arriba/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Vasculitis/fisiopatologíaRESUMEN
Conventional type 1 dendritic cells (cDC1) are critical regulators of anti-tumoral T-cell responses. The structure and abundance of intercellular contacts between cDC1 and CD8 T cells in cancer tissues is important to determine the outcome of the T-cell response. However, the molecular determinants controlling the stability of cDC1-CD8 interactions during cancer progression remain poorly investigated. Here, we generated a genetic model of non-small cell lung cancer crossed to a fluorescent cDC1 reporter (KP-XCR1venus) to allow the detection of cDC1-CD8T cell clusters in tumor tissues across tumor stages. We found that cDC1-CD8 clusters are abundant and productive at the early stages of tumor development but progressively diminish in advanced tumors. Transcriptional profiling and flow cytometry identified the adhesion molecule ALCAM/CD166 (Activated Leukocyte Cell Adhesion Molecule, ligand of CD6) as highly expressed by lung cDC1 and significantly downregulated in advanced tumors. Analysis of human datasets indicated that ALCAM is downregulated in non-small cell lung cancer and its expression correlates to better prognosis. Mechanistically, triggering ALCAM on lung cDC1 induces cytoskeletal remodeling and contact formation whereas its blockade prevents T-cell activation. Together, our results indicate that ALCAM is important to stabilize cDC1-CD8 interactions at early tumor stages, while its loss in advanced tumors contributes to immune evasion.
Asunto(s)
Antígenos CD , Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas , Células Dendríticas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Animales , Ratones , Antígenos CD/metabolismo , Antígenos CD/genética , Antígenos CD/inmunología , Proteínas Fetales/metabolismo , Proteínas Fetales/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Comunicación Celular/inmunología , Molécula de Adhesión Celular del Leucocito ActivadoRESUMEN
BACKGROUND: New drugs to tackle the next pathway or mutation fueling cancer are constantly proposed, but 97% of them are doomed to fail in clinical trials, largely because they are identified by cellular or in silico screens that cannot predict their in vivo effect. METHODS: We screened an Adeno-Associated Vector secretome library (> 1000 clones) directly in vivo in a mouse model of cancer and validated the therapeutic effect of the first hit, EMID2, in both orthotopic and genetic models of lung and pancreatic cancer. RESULTS: EMID2 overexpression inhibited both tumor growth and metastatic dissemination, consistent with prolonged survival of patients with high levels of EMID2 expression in the most aggressive human cancers. Mechanistically, EMID2 inhibited TGFß maturation and activation of cancer-associated fibroblasts, resulting in more elastic ECM and reduced levels of YAP in the nuclei of cancer cells. CONCLUSION: This is the first in vivo screening, precisely designed to identify proteins able to interfere with cancer cell invasiveness. EMID2 was selected as the most potent protein, in line with the emerging relevance of the tumor extracellular matrix in controlling cancer cell invasiveness and dissemination, which kills most of cancer patients.
Asunto(s)
Fibroblastos Asociados al Cáncer , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Núcleo Celular , Modelos Animales de Enfermedad , Detección Precoz del Cáncer , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Colágeno/metabolismoRESUMEN
A nephroblastoma is a tumor arising from metanephric blastema occurring in childhood. Among laboratory rodents, nephroblastoma has been frequently reported in rats, but it remains exceedingly rare in mice. The present work describes a nephroblastoma in a young mouse homozygous for the specific Trp53 R172H point mutation coupled with targeted deletion of the Pin1 gene. The affected kidney was effaced by a biphasic tumor with an epithelial component arranged in tubules surrounded by nests of blastemal cells. Immunohistochemically, the neoplasm was diffusely positive for Wilms' tumor antigen. The epithelial component expressed markers of renal tubular differentiation including wide-spectrum cytokeratin, E-cadherin and folate-binding protein. Furthermore, the neoplasm exhibited a high proliferative index and diffuse nucleocytoplasmic ß-catenin expression. Based on histological and immunohistochemical features, a diagnosis of nephroblastoma potentially associated with Trp53 loss and oncogenic ß-catenin activation has been proposed.
RESUMEN
Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.
Asunto(s)
Neoplasias de la Mama , Proteína p53 Supresora de Tumor , Femenino , Humanos , Aminoácidos/metabolismo , Aminoácidos Esenciales , Neoplasias de la Mama/patología , Glicina , Transportador de Aminoácidos Neutros Grandes 1/genética , Serina , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Giant cell tumor of bone (GCTB) is a common primary neoplasm of bone characterized by distinctive clinicopathological features. GCTB is exceedingly rare in nonhuman species, and it has been sporadically reported in cats, dogs, rats, and birds. This report describes a primary murine bone tumor that shares striking clinicopathological similarities with human GCTB. The neoplasm occurred in a 71-week-old C57BL/6 mouse heterozygous for the specific Trp53 R172H point mutation. Grossly, the tumor presented as a mono-ostotic nodular mass arising from the distal metaphysis of the right femur. Microscopically, the affected bone was effaced by an osteolytic neoplasm with focal infiltrations into the surrounding tissues. Similarly to what was reported for human GCTB, the murine neoplasm consisted of 3 main cell populations: (1) bundles of pleomorphic spindle-shaped mononuclear cells displaying an indefinite mesenchymal histogenesis with immunohistochemical expression of vimentin and smooth muscle actin, (2) scattered multinucleated giant cells exhibiting osteoclast differentiation with prominent tartrate-resistant acid phosphatase activity and immunoreactivity for monocyte/macrophage markers including CD45 and lysozyme, and (3) scattered round mononuclear cells consistent with activated macrophages and expressing CD45, lysozyme, and F4/80. Based on these morphological and immunohistological results, the murine bone tumor described in this study has been putatively classified as GCTB.
Asunto(s)
Neoplasias Óseas/genética , Tumor Óseo de Células Gigantes/genética , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/patología , Femenino , Tumor Óseo de Células Gigantes/diagnóstico , Tumor Óseo de Células Gigantes/patología , Células Gigantes , Inmunohistoquímica , Leucocitos Mononucleares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Mutación PuntualRESUMEN
Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.
Asunto(s)
Proliferación Celular , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/metabolismo , Factores Reguladores Miogénicos/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Transducción de Señal/fisiología , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Humanos , Factores de Transcripción MEF2 , Ratones , Proteína MioD/genética , Proteína MioD/metabolismo , Factores Reguladores Miogénicos/genética , Peptidilprolil Isomerasa de Interacción con NIMA , Péptidos/genética , Péptidos/metabolismo , Isomerasa de Peptidilprolil/genética , Fosforilación/fisiología , Estabilidad ProteicaRESUMEN
Chromatin organization plays a crucial role in tissue homeostasis. Heterochromatin relaxation and consequent unscheduled mobilization of transposable elements (TEs) are emerging as key contributors of aging and aging-related pathologies, including Alzheimer's disease (AD) and cancer. However, the mechanisms governing heterochromatin maintenance or its relaxation in pathological conditions remain poorly understood. Here we show that PIN1, the only phosphorylation-specific cis/trans prolyl isomerase, whose loss is associated with premature aging and AD, is essential to preserve heterochromatin. We demonstrate that this PIN1 function is conserved from Drosophila to humans and prevents TE mobilization-dependent neurodegeneration and cognitive defects. Mechanistically, PIN1 maintains nuclear type-B Lamin structure and anchoring function for heterochromatin protein 1α (HP1α). This mechanism prevents nuclear envelope alterations and heterochromatin relaxation under mechanical stress, which is a key contributor to aging-related pathologies.
Asunto(s)
Proteínas de Drosophila/metabolismo , Heterocromatina/metabolismo , Lamina Tipo B/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Estrés Mecánico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Células Cultivadas , Homólogo de la Proteína Chromobox 5/genética , Homólogo de la Proteína Chromobox 5/metabolismo , Elementos Transponibles de ADN/genética , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Humanos , Lamina Tipo B/química , Ratones , Ratones Endogámicos C57BL , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Neocórtex/citología , Neocórtex/metabolismo , Neuronas/citología , Neuronas/metabolismo , Membrana Nuclear/química , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Isomerasa de Peptidilprolil/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous beta-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-alpha2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation.
Asunto(s)
Secuencias AT-Hook , Núcleo Celular/metabolismo , Proteína HMGA2/química , Proteína HMGA2/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Aminoácidos Básicos/análisis , Animales , Línea Celular , Núcleo Celular/química , Cricetinae , Proteína HMGA2/análisis , Humanos , Ratones , Datos de Secuencia Molecular , Eliminación de Secuencia , Factores de Transcripción/análisis , alfa Carioferinas/metabolismoRESUMEN
Cellular choices are determined by developmental and environmental stimuli through integrated signal transduction pathways. These critically depend on attainment of proper activation levels that in turn rely on post-translational modifications (PTMs) of single pathway members. Among these PTMs, post-phosphorylation prolyl-isomerization mediated by PIN1 represents a unique mechanism of spatial, temporal and quantitative control of signal transduction. Indeed PIN1 was shown to be crucial for determining activation levels of several pathways and biological outcomes downstream to a plethora of stimuli. Of note, studies performed in different model organisms and humans have shown that hormonal, nutrient, and oncogenic stimuli simultaneously affect both PIN1 activity and the pathways that depend on PIN1-mediated prolyl-isomerization, suggesting the existence of evolutionarily conserved molecular circuitries centered on this isomerase. This review focuses on molecular mechanisms and cellular processes like proliferation, metabolism, and stem cell fate, that are regulated by PIN1 in physiological conditions, discussing how these are subverted in and hijacked by cancer cells. Current status and open questions regarding the use of PIN1 as biomarker and target for cancer therapy as well as clinical development of PIN1 inhibitors are also addressed.
RESUMEN
HMGA1 is an architectural transcription factor expressed at high levels in transformed cells and tumors. Several lines of evidence indicate that HMGA1 up-regulation is involved in the malignant transformation of thyroid epithelial cells. However, the mechanisms underlying the effect of HMGA1 on thyroid cancer cell phenotype are not fully understood. We now show that in thyroid cancer cells, HMGA1 down-regulation by small interfering RNA and antisense techniques results in enhanced transcriptional activity of p53, TAp63alpha, TAp73alpha, and, consequently, increased apoptosis. Coimmunoprecipitation and pull-down experiments with deletion mutants showed that the COOH-terminal oligomerization domain of p53 family members is required for direct interaction with HMGA1. Moreover, inhibition of HMGA1 expression in thyroid cancer cells resulted in increased p53 oligomerization in response to the DNA-damaging agent doxorubicin. Finally, electrophoretic mobility shift assay experiments showed that the p53-HMGA1 interaction results in reduced DNA-binding activity. These results indicate a new function of HMGA1 in the regulation of p53 family members, thus providing new mechanistic insights in tumor progression.
Asunto(s)
Proteína HMGA1a/metabolismo , Neoplasias de la Tiroides/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , ADN de Neoplasias/metabolismo , Proteína HMGA1a/biosíntesis , Proteína HMGA1a/deficiencia , Proteína HMGA1a/genética , Humanos , Inmunoprecipitación , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Activación Transcripcional , Transfección , Proteína p53 Supresora de Tumor/genética , Regulación hacia ArribaRESUMEN
The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120(E4F), interfering with p120(E4F) binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation.
Asunto(s)
Ciclina A/genética , Proteína HMGA2/metabolismo , Proteínas E4 de Adenovirus/química , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Ciclo Celular , Línea Celular , Transformación Celular Neoplásica , Cricetinae , ADN Complementario/genética , Proteína HMGA2/genética , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIH , Regiones Promotoras Genéticas , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Activación Transcripcional , Dedos de ZincRESUMEN
Mammary gland development, various stages of mammary tumorigenesis and breast cancer progression have the peptidyl-prolyl cis/trans isomerase PIN1 at their centerpiece, in virtue of the ability of this unique enzyme to fine-tune the dynamic crosstalk between multiple molecular pathways. PIN1 exerts its action by inducing conformational and functional changes on key cellular proteins, following proline-directed phosphorylation. Through this post-phosphorylation signal transduction mechanism, PIN1 controls the extent and direction of the cellular response to a variety of inputs, in physiology and disease. This review discusses PIN1's roles in normal mammary development and cancerous progression, as well as the clinical impact of targeting this enzyme in breast cancer patients.
Asunto(s)
Neoplasias de la Mama/patología , Glándulas Mamarias Humanas/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos , Factores de Transcripción E2F/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Femenino , Inestabilidad Genómica , Humanos , Glándulas Mamarias Humanas/crecimiento & desarrollo , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Transducción de SeñalRESUMEN
The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed in the majority of cancers and its activity strongly contributes to tumour initiation and progression. Inactivation of PIN1 function conversely curbs tumour growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening, we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinone-mimicking drug that generates reactive oxygen species and DNA damage, inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.
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Antineoplásicos/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Animales , Antineoplásicos/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatología , Ratones Desnudos , Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The HMGA architectural nuclear factors are involved in chromatin dynamics and their overexpression has been strongly linked to the neoplastic transformation process. Here we investigate the expression and post-translational modifications (PTMs) of HMGA proteins (HMGA1a, HMGA1b and HMGA2) in the rat prostatic cancer Dunning model (G, AT-1, and MAT-Ly-Lu cell lines). We demonstrate the expression of HMGA2, in addition to HMGA1a and HMGA1b, in both the anaplastic cell lines AT-1 and MAT-Ly-Lu and an extremely specific HMGA1a mono-methylation only in the most metastatic cell line MAT-Ly-Lu. The HMGA ectopic expression in HMGA-negative Dunning G cells does not significantly alter their growth ability, suggesting that, although HMGA expression is necessary for the progression of neoplastic transformation in several cellular models, in these cells it is not sufficient. These data suggest exploring HMGA2 as a potential marker in human prostate tumor and moreover indicate PTMs as an additional tool in the staging of tumor progression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas HMGA/biosíntesis , Neoplasias de la Próstata/metabolismo , Agar/química , Animales , Northern Blotting , Western Blotting , Línea Celular Tumoral , Cromatina/metabolismo , Cromatografía Liquida , ADN Complementario/metabolismo , Progresión de la Enfermedad , Humanos , Masculino , Espectrometría de Masas , Fenotipo , Plásmidos/metabolismo , Procesamiento Proteico-Postraduccional , Ratas , TransfecciónRESUMEN
BACKGROUND: The modest efficacy of available therapies for Hepatocellular carcinoma (HCC) indicates the need to develop novel therapeutic approaches. For the proteasome inhibitor Bortezomib (BZB), potentially attractive for HCC treatment, the mechanism of action is largely unknown. The BZB effect on E2Fs and the E2Fs control on the peptidylproline cis-trans isomerase (Pin1), prompted us to explore the BZB effect on the Pin1-E2F1 axis. METHODS: The tumorigenic cell line HuH7 together with the non-tumorigenic cells IHH and the human pluripotent stem cell derived hepatocytes (hPSC-H), were used as cellular models of HCC and normal liver cells, respectively. RESULTS: BZB reduces HuH7 growth as shown by cell counting, cell vitality test and cell cycle analysis; this is paralleled by the decrease of Pin1, E2F1, cyclin A2 and of the hyper-phosphorylated pRB. Pin1-E2F1 axis impairment justifies the anti-proliferative effect since Pin-E2F1 depletion decreases HuH7 growth while the over-expression rescues BZB-induced inhibition of proliferation. Moreover, Pin1-E2F1 promote HuH7 growth via the up-regulation of cyclin D1, cyclin E, cyclin A2, E2F2 and in part E2F3. Finally, in the control cells IHH and hPSC-H, BZB effect on cell vitality is not irrelevant, a fact correlated to the cellular proliferation rate. Thus, BZB effect on healthy liver tissue may not be entirely negligible hence caution should be exercised in its use in liver regeneration processes. CONCLUSION: For the first time we prove the functional involvement of the Pin1-E2F1 axis in the anti-proliferative effect of BZB indicating Pin1-E2F as an attractive target to control HCC cell growth.