RESUMEN
Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.
Asunto(s)
Aneuploidia , Neoplasias/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Mapeo Cromosómico , Cromosomas/genética , Factor de Transcripción E2F1/antagonistas & inhibidores , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Femenino , Biblioteca de Genes , Genómica , Humanos , Queratinas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Oncogenes , Sistemas de Lectura Abierta/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Nuclear pore complexes (NPCs) regulate nuclear-cytoplasmic transport, transcription, and genome integrity in eukaryotic cells. However, their functional roles in cancer remain poorly understood. We interrogated the evolutionary transcriptomic landscape of NPC components, nucleoporins (Nups), from primary to advanced metastatic human prostate cancer (PC). Focused loss-of-function genetic screen of top-upregulated Nups in aggressive PC models identified POM121 as a key contributor to PC aggressiveness. Mechanistically, POM121 promoted PC progression by enhancing importin-dependent nuclear transport of key oncogenic (E2F1, MYC) and PC-specific (AR-GATA2) transcription factors, uncovering a pharmacologically targetable axis that, when inhibited, decreased tumor growth, restored standard therapy efficacy, and improved survival in patient-derived pre-clinical models. Our studies molecularly establish a role of NPCs in PC progression and give a rationale for NPC-regulated nuclear import targeting as a therapeutic strategy for lethal PC. These findings may have implications for understanding how NPC deregulation contributes to the pathogenesis of other tumor types.
Asunto(s)
Factor de Transcripción E2F1/metabolismo , Glicoproteínas de Membrana/metabolismo , Poro Nuclear/fisiología , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Carcinogénesis , Núcleo Celular/metabolismo , Proliferación Celular , Factor de Transcripción GATA2/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Membrana Nuclear , Proteínas de Complejo Poro Nuclear , Transducción de SeñalRESUMEN
High levels of H2A.Z promote melanoma cell proliferation and correlate with poor prognosis. However, the role of the two distinct H2A.Z histone chaperone complexes SRCAP and P400-TIP60 in melanoma remains unclear. Here, we show that individual subunit depletion of SRCAP, P400, and VPS72 (YL1) results in not only the loss of H2A.Z deposition into chromatin but also a reduction of H4 acetylation in melanoma cells. This loss of H4 acetylation is particularly found at the promoters of cell cycle genes directly bound by H2A.Z and its chaperones, suggesting a coordinated regulation between H2A.Z deposition and H4 acetylation to promote their expression. Knockdown of each of the three subunits downregulates E2F1 and its targets, resulting in a cell cycle arrest akin to H2A.Z depletion. However, unlike H2A.Z deficiency, loss of the shared H2A.Z chaperone subunit YL1 induces apoptosis. Furthermore, YL1 is overexpressed in melanoma tissues, and its upregulation is associated with poor patient outcome. Together, these findings provide a rationale for future targeting of H2A.Z chaperones as an epigenetic strategy for melanoma treatment.
Asunto(s)
Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Histonas , Melanoma , Humanos , Melanoma/genética , Proliferación Celular/genética , Línea Celular Tumoral , Histonas/metabolismo , Histonas/genética , Acetilación , Apoptosis/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genéticaRESUMEN
The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.
Asunto(s)
Cromatina , Proteína de Retinoblastoma , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción AP-1/genéticaRESUMEN
The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.
Asunto(s)
División Celular , Movimiento Celular , Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica , Animales , Movimiento Celular/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , División Celular/genética , Mesodermo/metabolismo , Mesodermo/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/embriología , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/citología , Drosophila/genética , Drosophila/metabolismo , Drosophila/embriología , Factores de Transcripción de la Familia Snail/metabolismo , Factores de Transcripción de la Familia Snail/genéticaRESUMEN
Mitochondrial dysfunction causes poorly understood tissue-specific pathology stemming from primary defects in respiration, coupled with altered reactive oxygen species (ROS), metabolic signaling, and apoptosis. The A1555G mtDNA mutation that causes maternally inherited deafness disrupts mitochondrial ribosome function, in part, via increased methylation of the mitochondrial 12S rRNA by the methyltransferase mtTFB1. In patient-derived A1555G cells, we show that 12S rRNA hypermethylation causes ROS-dependent activation of AMP kinase and the proapoptotic nuclear transcription factor E2F1. This retrograde mitochondrial-stress relay is operative in vivo, as transgenic-mtTFB1 mice exhibit enhanced 12S rRNA methylation in multiple tissues, increased E2F1 and apoptosis in the stria vascularis and spiral ganglion neurons of the inner ear, and progressive E2F1-dependent hearing loss. This mouse mitochondrial disease model provides a robust platform for deciphering the complex tissue specificity of human mitochondrial-based disorders, as well as the precise pathogenic mechanism of maternally inherited deafness and its exacerbation by environmental factors.
Asunto(s)
Sordera/metabolismo , Modelos Animales de Enfermedad , Factor de Transcripción E2F1/metabolismo , Animales , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Oído Interno/patología , Ganglión/patología , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Neuronas/patología , ARN Ribosómico/metabolismo , Especies Reactivas de Oxígeno , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.
Asunto(s)
Ciclo Celular/genética , Ciclinas/genética , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F3/genética , Proteínas Ligasas SKP Cullina F-box/genética , Transcripción Genética , Línea Celular Tumoral , Ciclinas/metabolismo , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F2/metabolismo , Factor de Transcripción E2F3/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Aptitud Genética , Células HEK293 , Células HeLa , Humanos , Mutación , Osteoblastos/citología , Osteoblastos/metabolismo , Proteolisis , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.
Asunto(s)
Envejecimiento/fisiología , Ácido Ascórbico/farmacología , Diabetes Mellitus Experimental/prevención & control , Proteína de Retinoblastoma/metabolismo , Animales , Senescencia Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Factor de Transcripción E2F1/metabolismo , Desarrollo Embrionario/genética , Femenino , Fibroblastos/efectos de los fármacos , Técnicas de Sustitución del Gen , Células Secretoras de Insulina/patología , Ratones , Fosforilación , Embarazo , Proteína de Retinoblastoma/genética , Telómero/genéticaRESUMEN
The cell cycle depends on a sequence of steps that are triggered and terminated via the synthesis and degradation of phase-specific transcripts and proteins. Although much is known about how stage-specific transcription is activated, less is understood about how inappropriate gene expression is suppressed. Here, we demonstrate that Groucho, the Drosophila orthologue of TLE1 and other related human transcriptional corepressors, regulates normal cell cycle progression in vivo. We show that, although Groucho is expressed throughout the cell cycle, its activity is selectively inactivated by phosphorylation, except in S phase when it negatively regulates E2F1. Constitutive Groucho activity, as well as its depletion and the consequent derepression of e2f1, cause cell cycle phenotypes. Our results suggest that Cdk1 contributes to phase-specific phosphorylation of Groucho in vivo. We propose that Groucho and its orthologues play a role in the metazoan cell cycle that may explain the links between TLE corepressors and several types of human cancer.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Drosophila , Factor de Transcripción E2F1 , Proteínas Represoras , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ciclo Celular/genética , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Drosophila/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Fase G2 , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Fase S , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMEN
Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell-intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF-κB signaling and a transcription factor E2F1-dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL-induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine-driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F-metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Artritis Reumatoide/inmunología , Macrófagos/inmunología , Osteogénesis/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Factor de Transcripción E2F1/metabolismo , Femenino , Humanos , Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , FN-kappa B/metabolismo , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Although eukaryotic nuclei contain distinct architectural structures associated with noncoding RNAs (ncRNAs), their potential relationship to regulated transcriptional programs remains poorly understood. Here, we report that methylation/demethylation of Polycomb 2 protein (Pc2) controls relocation of growth-control genes between Polycomb bodies (PcGs) and interchromatin granules (ICGs) in response to growth signals. This movement is the consequence of binding of methylated and unmethylated Pc2 to the ncRNAs TUG1 and MALAT1/NEAT2, located in PcGs and ICGs, respectively. These ncRNAs mediate assembly of multiple corepressors/coactivators and can serve to switch mark recognition by "readers" of the histone code. Additionally, binding of NEAT2 to unmethylated Pc2 promotes E2F1 SUMOylation, leading to activation of the growth-control gene program. These observations delineate a molecular pathway linking the actions of subnuclear structure-specific ncRNAs and nonhistone protein methylation to relocation of transcription units in the three-dimensional space of the nucleus, thus achieving coordinated gene expression programs.
Asunto(s)
Núcleo Celular/metabolismo , Regulación de la Expresión Génica , ARN no Traducido/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Línea Celular , Cromatina/metabolismo , Factor de Transcripción E2F1/metabolismo , Células HeLa , Humanos , Ligasas , Metilación , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Proteínas del Grupo Polycomb , ARN Largo no Codificante , Proteínas Represoras/química , Sumoilación , Transcripción Genética , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process.
Asunto(s)
Proteínas de Drosophila , Neoplasias de la Próstata Resistentes a la Castración , Humanos , Animales , Femenino , Masculino , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Endorreduplicación , Ecdisona/genética , Ecdisona/metabolismo , Factor de Transcripción E2F1/genética , Factores de Transcripción/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMEN
Metastasis is the primary culprit behind cancer-related fatalities in multiple cancer types, including prostate cancer. Despite great advances, the precise mechanisms underlying prostate cancer metastasis are far from complete. By using a transgenic mouse prostate cancer model (TRAMP) with and without Phf8 knockout, we have identified a crucial role of PHF8 in prostate cancer metastasis. By complexing with E2F1, PHF8 transcriptionally upregulates SNAI1 in a demethylation-dependent manner. The upregulated SNAI1 subsequently enhances epithelial-to-mesenchymal transition (EMT) and metastasis. Given the role of the abnormally activated PHF8/E2F1-SNAI1 axis in prostate cancer metastasis and poor prognosis, the levels of PHF8 or the activity of this axis could serve as biomarkers for prostate cancer metastasis. Moreover, targeting this axis could become a potential therapeutic strategy for prostate cancer treatment. © 2024 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Factor de Transcripción E2F1 , Transición Epitelial-Mesenquimal , Histona Demetilasas , Neoplasias de la Próstata , Factores de Transcripción de la Familia Snail , Factores de Transcripción , Masculino , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/enzimología , Animales , Factores de Transcripción de la Familia Snail/metabolismo , Factores de Transcripción de la Familia Snail/genética , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Ratones , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Ratones Noqueados , Transducción de Señal , Metástasis de la Neoplasia , Ratones Transgénicos , Movimiento CelularRESUMEN
The mortality rate linked with nephrotic syndrome (NS) is quite high. The renal tubular injury influences the response of NS patients to steroid treatment. KN motif and ankyrin repeat domains 2 (KANK2) regulates actin polymerization, which is required for renal tubular cells to maintain their function. In this study, we found that the levels of KANK2 in patients with NS were considerably lower than those in healthy controls, especially in NS patients with acute kidney injury (AKI). To get a deeper understanding of the KANK2 transcriptional control mechanism, the core promoter region of the KANK2 gene was identified. KANK2 was further found to be positively regulated by E2F Transcription Factor 1 (E2F1), Transcription Factor AP-2 Gamma (TFAP2C), and Nuclear Respiratory Factor 1 (NRF1), both at mRNA and protein levels. Knocking down E2F1, TFAP2C, or NRF1 deformed the cytoskeleton of renal tubular cells and reduced F-actin content. EMSA and ChIP assays confirmed that all three transcription factors could bind to the upstream promoter transcription site of KANK2 to transactivate KANK2 in renal tubular epithelial cells. Our study suggests that E2F1, TFAP2C, and NRF1 play essential roles in regulating the KANK2 transcription, therefore shedding fresh light on the development of putative therapeutic options for the treatment of NS patients.
Asunto(s)
Síndrome Nefrótico , Factor Nuclear 1 de Respiración , Humanos , Factor Nuclear 1 de Respiración/metabolismo , Síndrome Nefrótico/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Factor de Transcripción E2F1/genética , Factor de Transcripción AP-2/genéticaRESUMEN
Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. Here, from an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), we identify a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; our genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. We demonstrate that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, we find that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, our study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation.
Asunto(s)
Células Madre Adultas/metabolismo , Proliferación Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factor de Transcripción E2F1/metabolismo , Intestinos/metabolismo , Prohibitinas/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular/fisiología , Homeostasis/fisiología , Interferencia de ARN/fisiología , Transducción de Señal/fisiologíaRESUMEN
ß-Arrestin 1 (ARRB1) has been recognized as a multifunctional adaptor protein in the last decade, beyond its original role in desensitizing G protein-coupled receptor signaling. Here, we identify that ARRB1 plays essential roles in mediating gastric cancer (GC) cell metabolism and proliferation, by combining cohort analysis and functional investigation using patient-derived preclinical models. Overexpression of ARRB1 was associated with poor outcome of GC patients and knockdown of ARRB1 impaired cell proliferation both ex vivo and in vivo. Intriguingly, ARRB1 depicted diverse subcellular localizations during a passage of organoid cultures (7 d) to exert dual functions. Further analysis revealed that nuclear ARRB1 binds with transcription factor E2F1 triggering up-regulation of proliferative genes, while cytoplasmic ARRB1 modulates metabolic flux by binding with the pyruvate kinase M2 isoform (PKM2) and hindering PKM2 tetramerization, which reduces pyruvate kinase activity and leads to cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis. As ARRB1 localization was shown mostly in the cytoplasm in human GC samples, therapeutic potential of the ARRB1-PKM2 axis was tested, and we found tumor proliferation could be attenuated by the PKM2 activator DASA-58, especially in ARRB1high organoids. Together, the data in our study highlight a spatiotemporally dependent role of ARRB1 in mediating GC cell metabolism and proliferation and implies reactivating PKM2 may be a promising therapeutic strategy in a subset of GC patients.
Asunto(s)
Piruvato Quinasa , Neoplasias Gástricas , beta-Arrestina 1 , Línea Celular Tumoral , Proliferación Celular/fisiología , Factor de Transcripción E2F1/metabolismo , Glucólisis/fisiología , Humanos , Isoformas de Proteínas/genética , Piruvato Quinasa/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismoRESUMEN
We have reported previously that during hypoxia exposure, the expression of mature miR-17â¼92 was first upregulated and then downregulated in pulmonary artery smooth muscle cells (PASMC) and in mouse lungs in vitro and in vivo. Here, we investigated the mechanisms regulating this biphasic expression of miR-17â¼92 in PASMC in hypoxia. We measured the level of primary miR-17â¼92 in PASMC during hypoxia exposure and found that short-term hypoxia exposure (3% O2, 6 h) induced the level of primary miR-17â¼92, whereas long-term hypoxia exposure (3% O2, 24 h) decreased its level, suggesting a biphasic regulation of miR-17â¼92 expression at the transcriptional level. We found that short-term hypoxia-induced upregulation of miR-17â¼92 was hypoxia-inducible factor 1α (HIF1α) and E2F1 dependent. Two HIF1α binding sites on miR-17â¼92 promoter were identified. We also found that long-term hypoxia-induced suppression of miR-17â¼92 expression could be restored by silencing of p53. Mutation of the p53-binding sites in the miR-17â¼92 promoter increased miR-17â¼92 promoter activity in both normoxia and hypoxia. Our findings suggest that the biphasic transcriptional regulation of miR-17â¼92 during hypoxia is controlled by HIF1/E2F1 and p53 in PASMC: during short-term hypoxia exposure, stabilization of HIF1 and induction of E2F1 induce the transcription of miR-17â¼92, whereas during long-term hypoxia exposure, hyperphosphorylation of p53 suppresses the expression of miR-17â¼92.NEW & NOTEWORTHY We showed that the biphasic transcriptional regulation of miR-17â¼92 during hypoxia is controlled by two distinct mechanisms: during short-term hypoxia exposure, induction of HIF1 and E2F1 upregulates miR-17â¼92. Longer hypoxia exposure induces hyperphosphorylation of p53 at ser15, which leads to its binding to miR-17â¼92 promoter and inhibition of its expression. Our findings provide novel insights into the spatiotemporal regulation of miR-17â¼92 that may play a role in the development of human lung diseases including pulmonary hypertension (PH).
Asunto(s)
Factor de Transcripción E2F1 , Subunidad alfa del Factor 1 Inducible por Hipoxia , MicroARNs , Arteria Pulmonar , Proteína p53 Supresora de Tumor , MicroARNs/genética , MicroARNs/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Fosforilación , Humanos , Animales , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Transcripción Genética , Hipoxia de la Célula/genética , Miocitos del Músculo Liso/metabolismo , Regiones Promotoras Genéticas/genética , Ratones , Hipoxia/metabolismo , Hipoxia/genética , Serina/metabolismo , Regulación de la Expresión Génica , Células CultivadasRESUMEN
Dysregulation of long noncoding RNA (lncRNA) expression plays a pivotal role in the initiation and progression of gastric cancer (GC). However, the regulation of lncRNA SNHG15 in GC has not been well studied. Mechanisms for ferroptosis by SNHG15 have not been revealed. Here, we aimed to explore SNHG15-mediated biological functions and underlying molecular mechanisms in GC. The novel SNHG15 was identified by analyzing RNA-sequencing (RNA-seq) data of GC tissues from our cohort and TCGA dataset, and further validated by qRT-PCR in GC cells and tissues. Gain- and loss-of-function assays were performed to examine the role of SNHG15 on GC both in vitro and in vivo. SNHG15 was highly expressed in GC. The enhanced SNHG15 was positively correlated with malignant stage and poor prognosis in GC patients. Gain- and loss-of-function studies showed that SNHG15 was required to affect GC cell growth, migration and invasion both in vitro and in vivo. Mechanistically, the oncogenic transcription factors E2F1 and MYC could bind to the SNHG15 promoter and enhance its expression. Meanwhile, SNHG15 increased E2F1 and MYC mRNA expression by sponging miR-24-3p. Notably, SNHG15 could also enhance the stability of SLC7A11 in the cytoplasm by competitively binding HNRNPA1. In addition, SNHG15 inhibited ferroptosis through an HNRNPA1-dependent regulation of SLC7A11/GPX4 axis. Our results support a novel model in which E2F1- and MYC-activated SNHG15 regulates ferroptosis via an HNRNPA1-dependent modulation of the SLC7A11/GPX4 axis, which serves as the critical effectors in GC progression, and provides a new therapeutic direction in the treatment of GC.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Progresión de la Enfermedad , Ferroptosis , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea A1 , Fosfolípido Hidroperóxido Glutatión Peroxidasa , ARN Largo no Codificante , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1/genética , Animales , Línea Celular Tumoral , Ratones , Ferroptosis/genética , Masculino , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Femenino , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Proliferación Celular/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Movimiento Celular/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Persona de Mediana Edad , Pronóstico , Ratones Desnudos , Transducción de Señal/genética , Retroalimentación FisiológicaRESUMEN
BACKGROUND: Bleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects. METHODS: Human lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model. RESULTS: Bleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin-induced mouse pulmonary fibrosis model. CONCLUSIONS: Our works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.
Asunto(s)
Bleomicina , Senescencia Celular , Reparación del ADN , Recombinasa Rad51 , Bleomicina/efectos adversos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Animales , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Humanos , Ratones , Reparación del ADN/efectos de los fármacos , Ratones Endogámicos C57BL , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Células A549 , Daño del ADN/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/efectos de los fármacosRESUMEN
Cryptorchidism is the most common urologic birth defect in men and is a predisposing factor of male infertility and testicular cancer, yet the etiology remains largely unknown. E2F1 microdeletions and microduplications contribute to cryptorchidism, infertility and testicular tumors. Although E2f1 deletion or overexpression in mice causes spermatogenic failure, the mechanism by which E2f1 influences testicular function is unknown. This investigation revealed that E2f1-null mice develop cryptorchidism with severe gubernacular defects and progressive loss of germ cells resulting in infertility and, in rare cases, testicular tumors. It was hypothesized that germ cell depletion resulted from an increase in WNT4 levels. To test this hypothesis, the phenotype of a double-null mouse model lacking both Wnt4 and E2f1 in germ cells was analyzed. Double-null mice are fertile. This finding indicates that germ cell maintenance is dependent on E2f1 repression of Wnt4, supporting a role for Wnt4 in germ cell survival. In the future, modulation of WNT4 expression in men with cryptorchidism and spermatogenic failure due to E2F1 copy number variations may provide a novel approach to improve their spermatogenesis and perhaps their fertility potential after orchidopexy.