RESUMEN
Respiratory diseases account for over 5 million deaths yearly and are a huge burden to healthcare systems worldwide. Murine models have been of paramount importance to decode human lung biology in vivo, but their genetic, anatomical, physiological and immunological differences with humans significantly hamper successful translation of research into clinical practice. Thus, to clearly understand human lung physiology, development, homeostasis and mechanistic dysregulation that may lead to disease, it is essential to develop models that accurately recreate the extraordinary complexity of the human pulmonary architecture and biology. Recent advances in micro-engineering technology and tissue engineering have allowed the development of more sophisticated models intending to bridge the gap between the native lung and its replicates in vitro Alongside advanced culture techniques, remarkable technological growth in downstream analyses has significantly increased the predictive power of human biology-based in vitro models by allowing capture and quantification of complex signals. Refined integrated multi-omics readouts could lead to an acceleration of the translational pipeline from in vitro experimental settings to drug development and clinical testing in the future. This review highlights the range and complexity of state-of-the-art lung models for different areas of the respiratory system, from nasal to large airways, small airways and alveoli, with consideration of various aspects of disease states and their potential applications, including pre-clinical drug testing. We explore how development of optimised physiologically relevant in vitro human lung models could accelerate the identification of novel therapeutics with increased potential to translate successfully from the bench to the patient's bedside.
Asunto(s)
Pulmón , Enfermedades Respiratorias , Humanos , Animales , Ratones , Pulmón/fisiología , Ingeniería de Tejidos/métodosRESUMEN
Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.
Asunto(s)
Colágeno/fisiología , Fibrosis Pulmonar/metabolismo , Biomarcadores , Células Cultivadas , Colágeno/química , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Factor 1 Inducible por Hipoxia , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Estrés Oxidativo/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-ß-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-ß-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Fibrosis Pulmonar Idiopática/fisiopatología , Movimiento Celular , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Femenino , Fibroblastos/metabolismo , Fibrosis/fisiopatología , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/patología , Masculino , Cultivo Primario de Células , Fibrosis Pulmonar/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma membrane, resulting in viral RNA genome delivery into the host. Despite ACE2's critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Células Epiteliales/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Chlorocebus aethiops , Exones , Células HEK293 , Humanos , Interferones/inmunología , Unión Proteica , Isoformas de Proteínas/genética , Sitios de Empalme de ARN , RNA-Seq , Sistema Respiratorio/citología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Transcriptoma , Regulación hacia Arriba , Células VeroRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease in which aging, environmental exposure(s) and genetic susceptibility have been implicated in disease pathogenesis, however, the causes and mechanisms of the progressive fibrotic cascade are still poorly understood. As epithelial-mesenchymal interactions are essential for normal wound healing, through human 2D and 3D in vitro studies, we tested the hypothesis that IPF fibroblasts (IPFFs) dysregulate alveolar epithelial homeostasis. Conditioned media from IPFFs exaggerated the wound-healing response of primary human Type II alveolar epithelial cells (AECs). Furthermore, AECs co-cultured with IPFFs exhibited irregular epithelialization compared with those co-cultured with control fibroblasts (NHLFs) or AECs alone, suggesting that epithelial homeostasis is dysregulated in IPF as a consequence of the abnormal secretory phenotype of IPFFs. Secretome analysis of IPFF conditioned media and functional studies identified the matricellular protein, SPARC, as a key mediator in the epithelial-mesenchymal paracrine signaling, with increased secretion of SPARC by IPFFs promoting persistent activation of alveolar epithelium via an integrin/focal adhesion/cellular-junction axis resulting in disruption of epithelial barrier integrity and increased macromolecular permeability. These findings suggest that in IPF fibroblast paracrine signaling promotes persistent alveolar epithelial activation, so preventing normal epithelial repair responses and restoration of tissue homeostasis. Furthermore, they identify SPARC-mediated paracrine signaling as a potential therapeutic target to promote the restoration of lung epithelial homoestasis in IPF patients.
RESUMEN
Idiopathic pulmonary fibrosis (IPF), the prototypic progressive fibrotic interstitial lung disease, is thought to be a consequence of repetitive micro-injuries to an ageing, susceptible alveolar epithelium. Ageing is a risk factor for IPF and incidence has been demonstrated to increase with age. Decreased (macro)autophagy with age has been reported extensively in a variety of systems and diseases, including IPF. However, it is undetermined whether the role of faulty autophagy is causal or coincidental in the context of IPF. Here, we report that in alveolar epithelial cells inhibition of autophagy promotes epithelial-mesenchymal transition (EMT), a process implicated in embryonic development, wound healing, cancer metastasis and fibrosis. We further demonstrate that this is attained, at least in part, by increased p62/SQSTM1 expression that promotes p65/RELA mediated-transactivation of an EMT transcription factor, Snail2 (SNAI2), which not only controls EMT but also regulates the production of locally acting profibrogenic mediators. Our data suggest that reduced autophagy induces EMT of alveolar epithelial cells and can contribute to fibrosis via aberrant epithelial-fibroblast crosstalk.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Fibrosis Pulmonar Idiopática/genética , Proteína Sequestosoma-1/genética , Factores de Transcripción de la Familia Snail/genética , Factor de Transcripción ReIA/genética , Células A549 , Envejecimiento/genética , Envejecimiento/patología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Autofagia/genética , Diferenciación Celular/genética , Fibroblastos/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Cultivo Primario de Células , Factores de Riesgo , Factores de TranscripciónRESUMEN
Development of biocompatible and functional scaffolds for tissue engineering is a major challenge, especially for development of polarised epithelia that are critical structures in tissue homeostasis. Different in vitro models of the lung epithelial barrier have been characterized using non-degradable polyethylene terephthalate membranes which limits their uses for tissue engineering. Although poly-L-lactic acid (PLLA) membranes are biodegradable, those prepared via conventional Diffusion Induced Phase Separation (DIPS) lack open-porous geometry and show limited permeability compromising their use for epithelial barrier studies. Here we used PLLA membranes prepared via a modification of the standard DIPS protocol to control the membrane surface morphology and permeability. These were bonded to cell culture inserts for use in barrier function studies. Pulmonary epithelial cells (H441) readily attached to the PLLA membranes and formed a confluent cell layer within two days. This was accompanied by a significant increase in trans-epithelial electrical resistance and correlated with the formation of tight junctions and vectorial cytokine secretion in response to TNFα. Our data suggest that a structurally polarized and functional epithelial barrier can be established on PLLA membranes produced via a non-standard DIPS protocol. Therefore, PLLA membranes have potential utility in lung tissue engineering applications requiring bio-absorbable membranes.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/fisiología , Pulmón/citología , Pulmón/fisiología , Poliésteres/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Implantes Absorbibles , Materiales Biocompatibles/química , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Línea Celular , Citocinas/metabolismo , Impedancia Eléctrica , Humanos , Ensayo de Materiales , Membranas Artificiales , Tereftalatos Polietilenos/química , Uniones Estrechas/fisiologíaRESUMEN
The contribution of epithelial-mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR-RAS-ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFß-induced profibrogenic responses in lung fibroblasts. This epithelial-mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.
Asunto(s)
Diferenciación Celular/genética , Fibrosis/genética , Enfermedades Respiratorias/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Línea Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Fibrosis/patología , Regulación de la Expresión Génica/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Miofibroblastos/metabolismo , Comunicación Paracrina/genética , Enfermedades Respiratorias/patologíaRESUMEN
Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.
Asunto(s)
Aminoácido Oxidorreductasas/antagonistas & inhibidores , Colágeno/química , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/química , Fibrosis Pulmonar/tratamiento farmacológico , Reticulina/química , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Aminoácidos/química , Animales , Fenómenos Biomecánicos , Estudios de Casos y Controles , Colágeno/metabolismo , Colágeno/ultraestructura , Reactivos de Enlaces Cruzados/química , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica , Homeostasis/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Mecanotransducción Celular , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/antagonistas & inhibidores , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Proteína-Lisina 6-Oxidasa , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Ratas , Ratas Sprague-Dawley , Reticulina/metabolismo , Reticulina/ultraestructura , Relación Estructura-Actividad , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Endometrial cancer is one of the most common gynaecological cancers in developed countries. Its incidence has increased 20% over the last decade and the death rate has increased >100% over the past two decades. Current models for prediction of prognosis and treatment response are suboptimal, and as such biomarkers to support clinical decision-making and contribute to individualised treatment are needed. In this study, we show that the E3-ubiquitin ligase PIR2/RNF144B is a potential targetable biomarker in endometrial cancer. At transcript level, it is expressed both in normal endometrium and tumour samples, but at protein level, it is expressed in tumours only. By using endometrial cancer cell lines, we demonstrated that PIR2/RNF144B is stabilised via phosphorylation downstream of GSK3ß and this is necessary for the proliferation of endometrial cancer cells, in the absence of oestrogenic growth stimuli. Here, inactivation of GSK3ß activity is associated with loss of PIR2/RNF144B protein and consequent inhibition of cell proliferation. Our results, therefore, substantiate PIR2/RNF144B as a novel candidate for targeted therapy in endometrial cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Endometriales/genética , Endometrio/metabolismo , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/genética , Ubiquitina-Proteína Ligasas/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Endometrio/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Cloruro de Litio/farmacología , Fosforilación , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.
Asunto(s)
Depsipéptidos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Acetilación , Biomarcadores , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Depsipéptidos/uso terapéutico , Epigénesis Genética , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/patología , MasculinoRESUMEN
Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham's F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.
Asunto(s)
Células A549/fisiología , Células Epiteliales Alveolares/fisiología , Diferenciación Celular/fisiología , Células A549/ultraestructura , Células Epiteliales Alveolares/ultraestructura , Técnicas de Cultivo de Célula , Ciclo Celular/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Microscopía Electrónica de Transmisión , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
The transcription factor p73 belongs to the p53 family of tumour suppressors and similar to other family members, transcribed as different isoforms with opposing pro- and anti-apoptotic functions. Unlike p53, p73 mutations are extremely rare in cancers. Instead, the pro-apoptotic activities of transcriptionally active p73 isoforms are commonly inhibited by over-expression of the dominant negative p73 isoforms. Therefore the relative ratio of different p73 isoforms is critical for the cellular response to a chemotherapeutic agent. Here, we analysed the expression of N-terminal p73 isoforms in cell lines and mouse tissues. Our data showed that the transcriptionally competent TAp73 isoform is abundantly expressed in cancer cell lines compared to the dominant negative ΔNp73 isoform. Interestingly, we detected higher levels of ΔNp73 in some mouse tissues, suggesting that ΔNp73 may have a physiological role in these tissues.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Empalme Alternativo , Animales , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Ratones , Proteínas Nucleares/genética , Isoformas de Proteínas , Interferencia de ARN , ARN Mensajero/metabolismo , Transcripción Genética , Transfección , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genéticaRESUMEN
p73 is a tumor suppressor belonging to the p53 family of transcription factors. Distinct isoforms are transcribed from the p73 locus. The use of 2 promoters at the N-terminus allows the expression of an isoform containing (TAp73) or not containing (ΔNp73) a complete N-terminal transactivation domain, with the latter isoform capable of a dominant negative effect over the former. In addition, both N-terminal variants are alternatively spliced at the C-terminus. TAp73 is a bona fide tumor suppressor, being able to induce cell death and cell cycle arrest; conversely, ΔNp73 shows oncogenic properties, inhibiting TAp73 and p53 functions. Here, we discuss the latest findings linking p73 to cancer. The generation of isoform specific null mice has helped in dissecting the contribution of TA versus ΔNp73 isoforms to tumorigenesis. The activity of both isoforms is regulated transcriptionally and by posttranslational modification. p73 dysfunction, particularly of TAp73, has been associated with mitotic abnormalities, which may lead to polyploidy and aneuploidy and thus contribute to tumorigenesis. Although p73 is only rarely mutated in cancer, the tumor suppressor actions of TAp73 are inhibited by mutant p53, a finding that has important implications for cancer therapy. Finally, we discuss the expression and role of p73 isoforms in human cancer, with a particular emphasis on the neuroblastoma cancer model. Broadly, the data support the hypothesis that the ratio between TAp73 and ΔNp73 is crucial for tumor progression and therapeutic response.
RESUMEN
p73, a transcription factor of the p53 family, plays a key role in many biological processes including neuronal development. Indeed, mice deficient for both TAp73 and ΔNp73 isoforms display neuronal pathologies, including hydrocephalus and hippocampal dysgenesis, with defects in the CA1-CA3 pyramidal cell layers and the dentate gyrus. TAp73 expression increases in parallel with neuronal differentiation and its ectopic expression induces neurite outgrowth and expression of neuronal markers in neuroblastoma cell lines and neural stem cells, suggesting that it has a pro-differentiation role. In contrast, ΔNp73 shows a survival function in mature cortical neurons as selective ΔNp73 null mice have reduced cortical thickness. Recent evidence has also suggested that p73 isoforms are deregulated in neurodegenerative pathologies such as Alzheimer's disease, with abnormal tau phosphorylation. Thus, in addition to its increasingly accepted contribution to tumorigenesis, the p73 subfamily also plays a role in neuronal development and neurodegeneration.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Sistema Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Diferenciación Celular , Proteínas de Unión al ADN/química , Humanos , Modelos Biológicos , Degeneración Nerviosa/metabolismo , Sistema Nervioso/patología , Proteínas Nucleares/química , Células Madre/citología , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/químicaRESUMEN
p73 is a p53-related transcription factor with fundamental roles in development and tumor suppression. Transcription from two different promoters on the p73 gene results in generation of transcriptionally active TAp73 isoforms and dominant negative DeltaNp73 isoforms with opposing pro- and anti-apoptotic functions. Therefore, the relative ratio of each isoform is an important determinant of the cell fate. Proteasomal degradation of p73 is mediated by polyubiquitination-dependent and -independent processes both of which appear, thus far, to lack selectivity for the TAp73 and DeltaNp73 isoforms. Here, we describe the characterization of another transcriptional target of TAp73; a ring finger domain ubiquitin ligase p73 Induced RING 2 protein (PIR2). Although PIR2 was initially identified a p53-induced gene (p53RFP), low abundance of PIR2 transcript in mouse embryonic fibroblasts of TAp73 KO mice compared with WT mice and comparison of PIR2 mRNA and protein levels following TAp73 or p53 overexpression substantiate TAp73 isoforms as strong inducers of PIR2. Although PIR2 expression was induced by DNA damage, its expression did not alter apoptotic response or cell cycle profile per se. However, coexpression of PIR2 with TAp73 or DeltaNp73 resulted in an increase of the TA/DeltaNp73 ratio, due to preferential degradation of DeltaNp73. Finally, PIR2 was able to relieve the inhibitory effect of DeltaNp73 on TAp73 induced apoptosis following DNA damage. These results suggest that PIR2, by being induced by TAp73 and degrading DeltaNp73, differentially regulates TAp73/DeltaNp73 stability, and, hence, it may offer a therapeutic approach to enhance the chemosensitivity of tumor cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Dominios RING Finger , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis , Daño del ADN , Células HCT116 , Humanos , Ratones , Unión Proteica , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteína Tumoral p73 , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
TAp63 belongs to the p53-tumour suppressor family and is capable of transactivating a set of target genes to induce cell cycle arrest and apoptosis. We showed that treatment of cancer cells with chemo-therapeutic drugs or the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) results in induction of TAp63 expression, which is in turn related with chemosensitivity. Indeed, induction of TAp63 by TSA affects sensitivity to chemo-therapeutic drugs via the cleavage of the trans-inhibitory domain of TAp63 by active caspases, resulting in generation of a transcriptionally hyper-active TAp63 fragment. Therefore therapeutic approaches that enhance TAp63 expression may offer an improvement in the management of chemoresistant tumours. In this study we tested the abilities of different HDAC inhibitors to induce TAp63 expression. We discovered that two HDAC inhibitors belonging to the hydroxamate group, namely TSA and LBH589, are the most efficient inducers of TAp63 expression. Finally, we found that induction of TAp63 expression in HCT116 cells depends on p53, as p53-negative HCT116 cells failed to induce significant TAp63 expression following treatment with different HDAC inhibitors.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Transactivadores/biosíntesis , Proteínas Supresoras de Tumor/biosíntesis , Línea Celular Tumoral , Humanos , Factores de TranscripciónRESUMEN
A new ionic Pd(II) complex, [(bipy)Pd(Pcurc)][CF(3)SO(3)], 1, with the metal center coordinated to two different chelating ligands, the pure curcumin (Pcurc) and the 4,4'-dinonyl-2,2'-bipyridine (bipy), has been synthesized, fully characterized, and its antitumoral mechanism and oxidant property have been investigated. The Pd(II) complex induces both cell growth inhibition and apoptosis of human prostate cancer cells, (LnCaP, PC3, and DU145) through the production of ROS and JNK phosphorylation associated with GSTp1 down-regulation. ROS production induced by complex 1 treatment activated apoptosis through mitochondrial membrane depolarization in all prostate cancer cells, with up-regulation of Bax and down-regulation of Bcl-2 proteins. In addition, while curcumin determines DNA damage and PARP cleavage, complex 1 does not elicit any activation of PARP enzyme. Taken together, these data validate the significance of curcumin complexation to a metal center and its conjugation to another functionalized bioactive ligand in the apoptosis signal transduction and enhancement of cell death in prostate cancer cell lines and suggest the potential of this design strategy in the improvement of the metal-based drugs cytotoxicity.