RESUMEN
Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Genes Homeobox , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Invasividad Neoplásica , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Transcripción Genética/genética , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
The A2A adenosine receptor (A2AR) is a G protein-coupled receptor and a major target of caffeine. The A2AR gene encodes alternative transcripts that are initiated from at least two independent promoters. The different transcripts of the A2AR gene contain the same coding region and 3'-untranslated region and different 5'-untranslated regions that are highly conserved among species. We report here that in addition to the production of the A2AR protein, translation from an upstream, out-of-frame AUG of the rat A2AR gene produces a 134-amino acid protein (designated uORF5). An anti-uORF5 antibody recognized a protein of the predicted size of uORF5 in PC12 cells and rat brains. Up-regulation of A2AR transcripts by hypoxia led to increased levels of both the A2AR and uORF5 proteins. Moreover, stimulation of A2AR increased the level of the uORF5 protein via post-transcriptional regulation. Expression of the uORF5 protein suppressed the AP1-mediated transcription promoted by nerve growth factor and modulated the expression of several proteins that were implicated in the MAPK pathway. Taken together, our results show that the rat A2AR gene encodes two distinct proteins (A2AR and uORF5) in an A2AR-dependent manner. Our study reveals a new example of the complexity of the mammalian genome and provides novel insights into the function of A2AR.
Asunto(s)
Regiones no Traducidas 5'/fisiología , Regulación de la Expresión Génica/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Sistemas de Lectura Abierta/fisiología , Receptor de Adenosina A2A/biosíntesis , Transducción de Señal/fisiología , Regiones no Traducidas 3'/fisiología , Animales , Hipoxia de la Célula/fisiología , Células HEK293 , Humanos , Células PC12 , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A2A/genéticaRESUMEN
Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Specifically, MYC-driven non-neuroendocrine SCLC is particularly resistant to standard therapies. Lurbinectedin was recently approved for the treatment of relapsed SCLC, but combinatorial approaches are needed to increase the depth and duration of responses to lurbinectedin. Using high-throughput screens, we found inhibitors of ataxia telangiectasia mutated and rad3 related (ATR) as the most effective agents for augmenting lurbinectedin efficacy. First-in-class ATR inhibitor berzosertib synergized with lurbinectedin in multiple SCLC cell lines, organoid, and in vivo models. Mechanistically, ATR inhibition abrogated S-phase arrest induced by lurbinectedin and forced cell cycle progression causing mitotic catastrophe and cell death. High CDKN1A/p21 expression was associated with decreased synergy due to G1 arrest, while increased levels of ERCC5/XPG were predictive of increased combination efficacy. Importantly, MYC-driven non-neuroendocrine tumors which are resistant to first-line therapies show reduced CDKN1A/p21 expression and increased ERCC5/XPG indicating they are primed for response to lurbinectedin-berzosertib combination. The combination is being assessed in a clinical trial NCT04802174.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Humanos , Recurrencia Local de Neoplasia , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas de la Ataxia Telangiectasia Mutada/metabolismoRESUMEN
Adenosine regulates important pathophysiological functions via four distinct adenosine receptor subtypes (A1, A2A, A2B, and A3). The A1 and A2A adenosine receptors (A1R and A2AR) are major targets of caffeine and have been extensively investigated. Huntington's disease (HD) is a dominant neurodegenerative disease caused by an abnormal CAG expansion in the Huntingtin gene. Since the first genetic HD model was created almost two decades ago, tremendous progress regarding the function of the adenosine receptors in HD has been made. Chronic intake of caffeine was recently shown to be positively associated with the disease onset of HD. Moreover, genetic polymorphism of A2AR is believed to impact the age of onset. Given the importance of adenosine receptors as drug targets for human diseases, this review highlights the recent findings that delineate the roles of adenosine receptors in HD and discusses their potential for serving as drug targets and/or biomarkers for HD. Adenosine is a purine nucleoside that regulates important physiological functions via four different adenosine receptors (A1, A2A, A2B, and A3). These adenosine receptors have seven transmembrane domains and belong to the G protein-coupled receptor family. The functions of the A1 adenosine receptor (A1R) and A2A adenosine receptor (A2AR) have been investigated relative to HD. In this review, we summarize the recent findings regarding the role of adenosine receptors in HD and discuss the potential application of adenosine receptors as drug targets and biomarkers for HD.
Asunto(s)
Enfermedad de Huntington/metabolismo , Receptores Purinérgicos P1/metabolismo , Animales , Autofagia , Cafeína/uso terapéutico , Humanos , Enfermedad de Huntington/complicaciones , Enfermedad de Huntington/tratamiento farmacológico , Enfermedades Metabólicas/etiología , Enfermedades Mitocondriales/etiología , Complejo de la Endopetidasa Proteasomal , Antagonistas de Receptores Purinérgicos P1/uso terapéuticoRESUMEN
Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases.
Asunto(s)
Neurobiología , Receptores Purinérgicos P1/fisiología , Animales , HumanosRESUMEN
BACKGROUND: Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A(2A) receptor (A(2A)R) regulates retinal waves and whether A(2A)R regulation of retinal waves acts via presynaptic SACs. METHODOLOGY/PRINCIPAL FINDINGS: We showed that A(2A)R was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A(2A)R decreased the frequency of spontaneous Ca²âº transients, suggesting that endogenous A(2A)R may up-regulate wave frequency. To investigate whether A(2A)R acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca²âº transient frequency was increased by expressing wild-type A(2A)R (A2AR-WT) in SACs, suggesting that A(2A)R may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A(2A)R-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A(2A)R may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A(2A)R mutant (A(2A)R-ΔC) in SACs, the wave frequency was reduced compared to the A(2A)R-WT, but was similar to the control, suggesting that the full-length A(2A)R in SACs is required for A(2A)R up-regulation of retinal waves. CONCLUSIONS/SIGNIFICANCE: A(2A)R up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full-length protein structure. Thus, by coupling with the downstream intracellular signaling, A(2A)R may have a great capacity to modulate patterned spontaneous activity during neural circuit refinement.
Asunto(s)
Potenciales de Acción , Células Amacrinas/citología , Receptor de Adenosina A2A/metabolismo , Retina/citología , Retina/crecimiento & desarrollo , Regulación hacia Arriba , Adenilil Ciclasas/metabolismo , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Imagen Molecular , Mutación , Ratas , Receptor de Adenosina A2A/química , Receptor de Adenosina A2A/deficiencia , Receptor de Adenosina A2A/genética , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Transducción de Señal , Potenciales SinápticosRESUMEN
The A2A adenosine receptor (A2AR) is a G-protein-coupled receptor. We previously reported that the C terminus of the A2AR binds to translin-associated protein X (TRAX) and modulates nerve growth factor (NGF)-evoked neurite outgrowth in PC12 cells. Herein, we show that neuritogenesis of primary hippocampal neurons requires p53 because blockage of p53 suppressed neurite outgrowth. The impaired neuritogenesis caused by p53 blockage was rescued by activation of the A2AR (designated the A2A rescue effect) in a TRAX-dependent manner. Importantly, suppression of a TRAX-interacting protein (kinesin heavy chain member 2A, KIF2A) inhibited the A2A rescue effect, whereas overexpression of KIF2A caused a rescue effect. Expression of a KIF2A fragment (KIF2A514), which disturbed the interaction between KIF2A and TRAX, blocked the rescue effect. Transient colocalization of TRAX and KIF2A was detected in the nucleus of PC12 cells upon NGF treatment. These data suggest that functional interaction between KIF2A and TRAX is critical for the A2A rescue effect. Moreover, p53 blockage during NGF treatment prevented the redistribution of KIF2A from the nucleus to the cytoplasmic region. Expression of a nuclear-retained KIF2A variant (NLS-KIF2A) did not rescue the impaired neurite outgrowth as did the wild-type KIF2A. Therefore, redistribution of KIF2A to the cytoplasmic fraction is a prerequisite for neurite outgrowth. Collectively, we demonstrate that KIF2A functions downstream of p53 to mediate neuritogenesis of primary hippocampal neurons and PC12 cells. Stimulation of the A2AR rescued neuritogenesis impaired by p53 blockage via an interaction between TRAX and KIF2A.