RESUMEN
mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.
Asunto(s)
Regulación de la Expresión Génica , Lipogénesis , Procesamiento Postranscripcional del ARN , Transducción de Señal , Animales , Núcleo Celular/metabolismo , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Femenino , Xenoinjertos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismoRESUMEN
Kidney cyst expansion in tuberous sclerosis complex (TSC) or polycystic kidney disease (PKD) requires active secretion of chloride (Cl-) into the cyst lumen. In PKD, Cl- secretion is primarily mediated via the cystic fibrosis transmembrane conductance regulator (CFTR) in principal cells. Kidney cystogenesis in TSC is predominantly composed of type A intercalated cells, which do not exhibit noticeable expression of CFTR. The identity of the Cl--secreting molecule(s) in TSC cyst epithelia remains speculative. RNA-sequencing analysis results were used to examine the expression of FOXi1, the chief regulator of acid base transporters in intercalated cells, along with localization of Cl- channel 5 (ClC5), in various models of TSC. Results from Tsc2+/- mice showed that the expansion of kidney cysts corresponded to the induction of Foxi1 and correlated with the appearance of ClC5 and H+-ATPase on the apical membrane of cyst epithelia. In various mouse models of TSC, Foxi1 was robustly induced in the kidney, and ClC5 and H+-ATPase were expressed on the apical membrane of cyst epithelia. Expression of ClC5 was also detected on the apical membrane of cyst epithelia in humans with TSC but was absent in humans with autosomal dominant PKD or in a mouse model of PKD. These results indicate that ClC5 is expressed on the apical membrane of cyst epithelia and is a likely candidate mediating Cl- secretion into the kidney cyst lumen in TSC.
Asunto(s)
Quistes , Enfermedades Renales Poliquísticas , Esclerosis Tuberosa , Humanos , Animales , Ratones , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Cloruros/metabolismo , Esclerosis Tuberosa/metabolismo , Riñón/metabolismo , Epitelio/metabolismo , Factores de Transcripción Forkhead/metabolismoRESUMEN
Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H+-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl-/H+ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H+-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H+-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H+-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.
Asunto(s)
Anhidrasa Carbónica II/genética , Factores de Transcripción Forkhead/genética , Insuficiencia Renal/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Animales , Quistes/genética , Quistes/patología , Modelos Animales de Enfermedad , Humanos , Riñón/metabolismo , Riñón/patología , Ratones , Mutación/genética , ATPasas de Translocación de Protón/genética , Insuficiencia Renal/patología , Canales Catiónicos TRPP/genética , Esclerosis TuberosaRESUMEN
The mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates multiple processes, including gene transcription, protein synthesis, ribosome biogenesis, autophagy, cell metabolism, and cell growth [...].
Asunto(s)
Transducción de Señal , Serina-Treonina Quinasas TOR , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transducción de Señal/genética , Biosíntesis de Proteínas , AutofagiaRESUMEN
Rationale: Lymphangioleiomyomatosis (LAM) is a metastatic neoplasm of reproductive-age women associated with mutations in tuberous sclerosis complex genes. LAM causes cystic remodeling of the lung and progressive respiratory failure. The sources and cellular characteristics of LAM cells underlying disease pathogenesis remain elusive.Objectives: Identification and characterization of LAM cells in human lung and uterus using a single-cell approach.Methods: Single-cell and single-nuclei RNA sequencing on LAM (n = 4) and control (n = 7) lungs, immunofluorescence confocal microscopy, ELISA, and aptamer proteomics were used to identify and validate LAMCORE cells and secreted biomarkers, predict cellular origins, and define molecular and cellular networks in LAM.Measurements and Main Results: A unique cell type termed LAMCORE was identified, which was distinct from, but closely related to, lung mesenchymal cells. LAMCORE cells expressing signature genes included known LAM markers such as PMEL, FIGF, CTSK, and MLANA and novel biomarkers validated by aptamer screening, ELISA, and immunofluorescence microscopy. LAM cells in lung and uterus are morphologically indistinguishable and share similar gene expression profiles and biallelic TSC2 mutations, supporting a potential uterine origin for the LAMCORE cell. Effects of LAM on resident pulmonary cell types indicated recruitment and activation of lymphatic endothelial cells.Conclusions: A unique population of LAMCORE cells was identified in lung and uterus of patients with LAM, sharing close transcriptomic identity. LAM cell selective markers, secreted biomarkers, and the predicted cellular molecular features provide new insights into the signaling and transcriptional programs that may serve as diagnostic markers and therapeutic targets to influence the pathogenesis of LAM.
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Biomarcadores de Tumor/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Linfangioleiomiomatosis/diagnóstico , Linfangioleiomiomatosis/genética , Transcriptoma/genética , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/genética , Adulto , Anciano , Femenino , Humanos , Persona de Mediana Edad , Análisis de la Célula Individual , Estados UnidosRESUMEN
In the connectivity map (CMap) approach to drug repositioning and development, transcriptional signature of disease is constructed by differential gene expression analysis between the diseased tissue or cells and the control. The negative correlation between the transcriptional disease signature and the transcriptional signature of the drug, or a bioactive compound, is assumed to indicate its ability to "reverse" the disease process. A major limitation of traditional CMaP analysis is the use of signatures derived from bulk disease tissues. Since the key driver pathways are most likely dysregulated in only a subset of cells, the "averaged" transcriptional signatures resulting from bulk analysis lack the resolution to effectively identify effective therapeutic agents. The use of single-cell RNA-seq (scRNA-seq) transcriptomic assay facilitates construction of disease signatures that are specific to individual cell types, but methods for using scRNA-seq data in the context of CMaP analysis are lacking. Lymphangioleiomyomatosis (LAM) mutations in TSC1 or TSC2 genes result in the activation of the mTOR complex 1 (mTORC1). The mTORC1 inhibitor Sirolimus is the only FDA-approved drug to treat LAM. Novel therapies for LAM are urgently needed as the disease recurs with discontinuation of the treatment and some patients are insensitive to the drug. We developed methods for constructing disease transcriptional signatures and CMaP analysis using scRNA-seq profiling and applied them in the analysis of scRNA-seq data of lung tissue from naïve and sirolimus-treated LAM patients. New methods successfully implicated mTORC1 inhibitors, including Sirolimus, as capable of reverting the LAM transcriptional signatures. The CMaP analysis mimicking standard bulk-tissue approach failed to detect any connection between the LAM signature and mTORC1 signaling. This indicates that the precise signature derived from scRNA-seq data using our methods is the crucial difference between the success and the failure to identify effective therapeutic treatments in CMaP analysis.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Conectoma/métodos , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Linfangioleiomiomatosis/patología , Análisis de la Célula Individual/métodos , Serina-Treonina Quinasas TOR/metabolismo , Antibióticos Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/metabolismo , Pronóstico , Análisis de Secuencia de ARN , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Tumor-associated macrophages (TAMs), key immune cells in the tumor microenvironment, are shown to be closely correlated with the progression of non-small cell lung cancer (NSCLC). Cancer stem cells (CSCs) can contribute to NSCLC progression as well. We aimed to clarify whether TAMs promote the progression of NSCLC by mainly affecting the activities of CSCs. We found that TAM-like cells promoted CSC-like properties in NSCLC cells in vitro, which was mediated by TAM-derived IL-10. TAM-derived IL-10 promoted CSC-like properties of NSCLC cells through JAK1/STAT1/NF-κB/Notch1 signaling. Blockade of IL-10/JAK1 signaling inhibited TAM-mediated NSCLC tumor growth in vivo, and the TAM-mediated expression of CSC-related and mesenchymal-related genes in NSCLC. Lastly, expression levels of these signaling molecules were significantly correlated with survival of NSCLC patients. Therefore, IL-10/JAK1 signaling might be a potential therapeutic target for NSCLC treatment.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Interleucina-10/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Macrófagos/metabolismo , Células Madre Neoplásicas/metabolismo , Transducción de Señal/fisiología , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Femenino , Humanos , Janus Quinasa 1/metabolismo , Macrófagos/patología , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Células Madre Neoplásicas/patología , Receptor Notch1/metabolismo , Factor de Transcripción STAT1/metabolismo , Microambiente Tumoral/fisiologíaRESUMEN
BACKGROUND/OBJECTIVE: The Active Healthy Kids 2018 Hong Kong Report Card provides evidence-based assessment across 12 indicators of physical activity behaviors, sleep, and related community and government initiatives for children and youth. METHODS: The systematic development process provided by the Active Healthy Kids Global Alliance was used. The best available data from the past 10 years were reviewed by a panel of experts. According to predefined benchmarks, letter grades were assigned to 12 indicators (Overall Physical Activity, Organized Sport Participation, Active Play, Active Transportation, Sedentary Behaviors, Physical Fitness, Sleep, Family, School, Community and Environment, Government, and Obesity). RESULTS: Three of the five activity behaviors received C- or C grades: Overall Physical Activity (C-), Sedentary Behaviors (C-), and Organized Sport Participation (C). Active Transportation was graded B + . Same to the 2016 Hong Kong Report Card, Active Play could not be graded. School and Government were graded C. Family and Community and Environment was graded D- and B, respectively. Three new indicators were added after the 2016 Report Card and they were graded from C- (Sleep) to D (Physical Fitness) or D- (Obesity). CONCLUSIONS: Children and youth in Hong Kong have low physical activity and physical fitness levels and high sedentary behaviors despite a generally favorable community environment. A high prevalence of obesity and low levels of family support warrant more public health action. Researchers should address the surveillance gap in active play and peer support.
RESUMEN
Genetic alterations drive metabolic reprograming to meet increased biosynthetic precursor and energy demands for cancer cell proliferation and survival in unfavorable environments. A systematic study of gene-metabolite regulatory networks and metabolic dysregulation should reveal the molecular mechanisms underlying prostate cancer (PCa) pathogenesis. Herein, we performed gas chromatography-mass spectrometry (GC-MS)-based metabolomics and RNA-seq analyses in prostate tumors and matched adjacent normal tissues (ANTs) to elucidate the molecular alterations and potential underlying regulatory mechanisms in PCa. Significant accumulation of metabolic intermediates and enrichment of genes in the tricarboxylic acid (TCA) cycle were observed in tumor tissues, indicating TCA cycle hyperactivation in PCa tissues. In addition, the levels of fumarate and malate were highly correlated with the Gleason score, tumor stage and expression of genes encoding related enzymes and were significantly related to the expression of genes involved in branched chain amino acid degradation. Using an integrated omics approach, we further revealed the potential anaplerotic routes from pyruvate, glutamine catabolism and branched chain amino acid (BCAA) degradation contributing to replenishing metabolites for TCA cycle. Integrated omics techniques enable the performance of network-based analyses to gain a comprehensive and in-depth understanding of PCa pathophysiology and may facilitate the development of new and effective therapeutic strategies.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Metabolómica/métodos , Neoplasias de la Próstata/patología , Ciclo del Ácido Cítrico , Fumaratos/análisis , Cromatografía de Gases y Espectrometría de Masas , Regulación Neoplásica de la Expresión Génica , Humanos , Malatos/análisis , Masculino , Clasificación del Tumor , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Análisis de Secuencia de ARNRESUMEN
OBJECTIVES: To determine the characteristics and effectiveness of motor skill interventions in children with developmental coordination disorder (DCD) and to identify potential moderators of training effects using meta-analysis. DATA SOURCES: A search was conducted in 6 databases (CINAHL Plus, Cochrane Library, Embase, ERIC, PsycINFO, and PubMed) for articles published between 1995 and August 2017 using search items which were grouped into 3 components (motor skill interventions, DCD, and age group of interest). STUDY SELECTION: Studies were included if they recruited children 3 to 17 years of age with DCD, reported performance of motor-related skills as outcomes, were published in peer-reviewed journals, and were written in English. Qualitative synthesis was conducted for all included studies. Quantitative synthesis (meta-analysis) was only conducted for studies using a (quasi) randomized controlled trial design. DATA EXTRACTION: Methodology, participant characteristics, intervention components, outcomes, and statistically significant training effects of each included study were extracted. DATA SYNTHESIS: Sixty-six studies met the inclusion criteria with 18 of the studies eligible for meta-analysis. Motor performance and cognitive, emotional, and other psychological factors were the most common outcomes. Other 3 outcome categories included perceptions and/or satisfaction regarding the children's improvement from significant others, physical fitness, and physical activity and participation. Immediate and moderate training effects were found for motor performance (Hedges g=.63; 95% confidence interval [CI], .31-.94; P<.001) and cognitive, emotional, and other psychological factors (Hedges g=0.65; 95% CI, 0.25-1.04; P=.001). Additionally, dose (minutes in total) and frequency of the intervention were significant moderators of training effect on motor performance. CONCLUSIONS: Motor skill interventions are effective in improving motor competence and performance on cognitive, emotional, and other psychological aspects in children with DCD in the short term. These effects are more robust in interventions using a large training dose and a practicing schedule of high frequency.
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Trastornos de la Destreza Motora/rehabilitación , Destreza Motora , Modalidades de Fisioterapia , Desempeño Psicomotor , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Resultado del TratamientoRESUMEN
The mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals from growth factors, nutrients, and cellular energy status to control a wide range of metabolic processes, including mRNA biogenesis; protein, nucleotide, and lipid synthesis; and autophagy. Deregulation of the mTORC1 pathway is found in cancer as well as genetic disorders such as tuberous sclerosis complex (TSC) and sporadic lymphangioleiomyomatosis. Recent studies have shown that the mTORC1 inhibitor rapamycin and its analogs generally suppress proliferation rather than induce apoptosis. Therefore, it is critical to use alternative strategies to induce death of cells with activated mTORC1. In this study, a small-molecule screen has revealed that the combination of glutaminase (GLS) and heat shock protein 90 (Hsp90) inhibitors selectively triggers death of TSC2-deficient cells. At a mechanistic level, high mTORC1-driven translation rates in TSC1/2-deficient cells, unlike wild-type cells, sensitizes these cells to endoplasmic reticulum (ER) stress. Thus, Hsp90 inhibition drives accumulation of unfolded protein and ER stress. When combining proteotoxic stress with oxidative stress by depletion of the intracellular antioxidant glutathione by GLS inhibition, acute cell death is observed in cells with activated mTORC1 signaling. This study suggests that this combination strategy may have the potential to be developed into a therapeutic use for the treatment of mTORC1-driven tumors.
Asunto(s)
Glutaminasa/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Animales , Apoptosis/efectos de los fármacos , Benzoquinonas/farmacología , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Glutamato Deshidrogenasa/antagonistas & inhibidores , Glutamato Deshidrogenasa/metabolismo , Glutaminasa/metabolismo , Glutamina/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Modelos Biológicos , Oxidación-Reducción/efectos de los fármacos , Fenotipo , Sirolimus/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfuros/farmacología , Tiadiazoles/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The benefits of physical activity (PA) for children with disabilities are well documented, and children with physical disabilities (PD) are often less active than peers with other disability types. Various correlates associated with PA in children with PD have been identified in separate studies, and a thorough analysis of these correlates could aid in understanding and designing interventions that promote children with PD to be more physically active. The purpose of this systematic review was to provide a comprehensive summary of the correlates of PA in children with PD. METHOD: A systematic search using PubMed, CINAHL, Cochrane Library, PsychINFO, Eric, and EMbase was initiated in October 2014 to identify studies examining the correlates of PA in children with PD aged 6-18years. Two researchers independently screened studies, assessed their methodological quality, and extracted relevant data. The correlates were synthesized and further assessed semi-quantitatively. RESULTS: A total of 45 articles were included in the detailed review. Several modifiable physical, psychological, and environmental correlates were consistently and positively associated with PA in children with PD. Some non-modifiable correlates (e.g., intellectual ability, parents' ethnicity) were found to be consistently and negatively associated with PA. CONCLUSIONS: The correlates of PA in children with PD are multifaceted and along many dimensions. This review can have implications for future studies and these may confirm the consistency of variables related to PA. Insights derived from the outcomes may also foster the measurement of the magnitude of associations that could assist the development of future interventions.
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Niños con Discapacidad , Ejercicio Físico/fisiología , Actividades Recreativas , Adolescente , Niño , HumanosRESUMEN
Lymphangioleiomyomatosis (LAM) is a destructive lung disease of women associated with the metastasis of tuberin-null cells with hyperactive mammalian target of rapamycin complex 1 (mTORC1) activity. Clinical trials with the mTORC1 inhibitor rapamycin have revealed partial efficacy but are not curative. Pregnancy appears to exacerbate LAM, suggesting that estrogen (E2) may play a role in the unique features of LAM. Using a LAM patient-derived cell line (bearing biallelic Tuberin inactivation), we demonstrate that E2 stimulates a robust and biphasic activation of ERK2 and transcription of the late response-gene Fra1 associated with epithelial-to-mesenchymal transition. In a carefully orchestrated collaboration, activated mTORC1/S6K1 signaling enhances the efficiency of Fra1 translation of Fra1 mRNA transcribed by the E2-ERK2 pathway, through the phosphorylation of the S6K1-dependent eukaryotic translation initiation factor 4B. Our results indicate that targeting the E2-ERK pathway in combination with the mTORC1 pathway may be an effective combination therapy for LAM.
Asunto(s)
Estradiol/metabolismo , Linfangioleiomiomatosis/etiología , Linfangioleiomiomatosis/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal , Factores Eucarióticos de Iniciación/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Linfangioleiomiomatosis/genética , Sistema de Señalización de MAP Quinasas , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Invasividad Neoplásica , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Lymphangioleiomyomatosis (LAM) is a rare neoplastic metastatic disease affecting women of childbearing age. LAM is caused by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) as a consequence of tuberous sclerosis complex (TSC) 1/2 inactivation. Clinically, LAM results in cystic lung destruction. mTORC1 inhibition using rapamycin analogs (rapalogs) is partially effective in reducing disease progression and improving lung function. However, cessation of treatment results in continued progression of the disease. In the present study, we investigated the effectiveness of the combination of rapamycin treatment with resveratrol, an autophagy inhibitor, in the TSC2-null xenograft tumor model. We determined that this combination inhibits phosphatidylinositol-4,5-bisphosphate 3-kinase PI3K/Akt/mTORC1 signaling and activates apoptosis. Therefore, the combination of rapamycin and resveratrol may be an effective clinical strategy for treatment of LAM and other diseases with mTORC1 hyperactivation.
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Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Linfangioleiomiomatosis/tratamiento farmacológico , Sirolimus/farmacología , Estilbenos/farmacología , Proteínas Supresoras de Tumor/genética , Neoplasias Uterinas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Quimioterapia Combinada , Femenino , Humanos , Linfangioleiomiomatosis/genética , Linfangioleiomiomatosis/inmunología , Linfangioleiomiomatosis/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones SCID , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Complejos Multiproteicos/inmunología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Ratas , Resveratrol , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/inmunología , Resultado del Tratamiento , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/inmunología , Neoplasias Uterinas/genética , Neoplasias Uterinas/inmunología , Neoplasias Uterinas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lymphangioleiomyomatosis (LAM) is a female-predominant cystic lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis complex 2 (TSC2) mutations and mammalian target of rapamycin (mTOR) complex (mTORC) 1 activation. Clinical response to the mTORC1 inhibitors has been limited, prompting a search for additional therapy for LAM. In this study, we investigated the impact of TSC2 on the expression of poly (ADP-ribose) polymerase (PARP)-1 that initiates the DNA repair pathway, and tested the efficacy of PARP1 inhibitors in the survival of TSC2-deficient (TSC2(-)) cells. We analyzed publicly available expression arrays of TSC2(-) cells and validated the findings using real-time RT-PCR, immunoblotting, and immunohistochemistry. We examined the impact of rapamycin and Torin 1 on PARP1 expression. We also tested the effect of PARP1 inhibitors, 8-hydroxy-2-methylquinazoline-4-one and 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline, on the survival of TSC2(-) cells. We identified the up-regulation of PARP1 in TSC2(-) cells relative to cells in which wild-type TSC2 has been reintroduced (TSC2-addback [TSC2(+)] cells). The transcript levels of PARP1 in TSC2(-) cells were not affected by rapamycin. PARP1 levels were increased in TSC2(-) cells, xenograft tumors of rat-derived TSC2(-) cells, renal cystadenomas from Tsc2(+/-) mice, and human LAM nodules. RNA interference of mTOR failed to reduce PARP1 levels. Proliferation and survival of TSC2(-) cells was reduced in response to PARP1 inhibitor treatment, more so than TSC2(+) cells. TSC2(-) cells exhibit higher levels of PARP1 relative to TSC2(+) cells in an mTOR-insensitive manner. PARP1 inhibitors selectively suppress the growth and induce apoptosis of TSC2(-) cells from patients with LAM. Targeting PARP1 may be beneficial in the treatment of LAM and other neoplasm with mTORC1 activation.
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Neoplasias Pulmonares/enzimología , Linfangioleiomiomatosis/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Quinazolinas/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular , Reparación del ADN , Resistencia a Antineoplásicos , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Isoquinolinas/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Linfangioleiomiomatosis/tratamiento farmacológico , Ratones Endogámicos C57BL , Ratones SCID , Terapia Molecular Dirigida , Ftalazinas/farmacología , Piperazinas/farmacología , Piperidinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Ratas , Sirolimus/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome characterized by benign tumors in multiple organs, including the brain and kidney. TSC-associated tumors exhibit hyperactivation of mammalian target of rapamycin complex 1 (mTORC1), a direct inhibitor of autophagy. Autophagy can either promote or inhibit tumorigenesis, depending on the cellular context. The role of autophagy in the pathogenesis and treatment of the multisystem manifestations of TSC is unknown. We found that the combination of mTORC1 and autophagy inhibition was more effective than either treatment alone in inhibiting the survival of tuberin (TSC2)-null cells, growth of TSC2-null xenograft tumors, and development of spontaneous renal tumors in Tsc2(+/-) mice. Down-regulation of Atg5 induced extensive central necrosis in TSC2-null xenograft tumors, and loss of one allele of Beclin1 almost completely blocked macroscopic renal tumor formation in Tsc2(+/-) mice. Surprisingly, given the finding that lowering autophagy blocks TSC tumorigenesis, genetic down-regulation of p62/sequestosome 1 (SQSTM1), the autophagy substrate that accumulates in TSC tumors as a consequence of low autophagy levels, strongly inhibited the growth of TSC2-null xenograft tumors. These data demonstrate that autophagy is a critical component of TSC tumorigenesis, suggest that mTORC1 inhibitors may have autophagy-dependent prosurvival effects in TSC, and reveal two distinct therapeutic targets for TSC: autophagy and the autophagy target p62/SQSTM1.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Choque Térmico/metabolismo , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Autofagia/genética , Autofagia/fisiología , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Genes p53 , Proteínas de Choque Térmico/genética , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Complejos Multiproteicos , Proteínas/genética , Proteínas/metabolismo , Proteína Sequestosoma-1 , Serina-Treonina Quinasas TOR , Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.
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Diferenciación Celular , Osteoclastos , Fibrosis Pulmonar , Dióxido de Silicio , Silicosis , Dióxido de Silicio/toxicidad , Animales , Humanos , Osteoclastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/metabolismo , Ratones , Silicosis/patología , Silicosis/metabolismo , Silicosis/etiología , Diferenciación Celular/efectos de los fármacos , Ligando RANK/metabolismo , Modelos Animales de Enfermedad , Masculino , Pulmón/patología , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Macrófagos Alveolares/efectos de los fármacos , FemeninoRESUMEN
Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)-2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)-2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.
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Estradiol/análogos & derivados , Estradiol/efectos adversos , Matriz Extracelular/efectos de los fármacos , Neoplasias Pulmonares/secundario , Linfangioleiomiomatosis/patología , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Antineoplásicos/farmacología , Colágeno Tipo IV/metabolismo , Evaluación Preclínica de Medicamentos , Estradiol/farmacología , Matriz Extracelular/metabolismo , Femenino , Fulvestrant , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Linfangioleiomiomatosis/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones SCID , Ratas , Receptores de Estradiol/antagonistas & inhibidores , Análisis de Supervivencia , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease that predominantly affects women. LAM cells carry TSC1/TSC2 mutations, causing mTORC1 hyperactivation and uncontrolled cell growth. mTORC1 inhibitors stabilize lung function; however, sustained efficacy requires long-term administration, and some patients fail to tolerate or respond to therapy. Although the genetic basis of LAM is known, mechanisms underlying LAM pathogenesis remain elusive. We integrated single-cell RNA sequencing and single-nuclei ATAC-seq of LAM lungs to construct a gene regulatory network controlling the transcriptional program of LAM cells. We identified activation of uterine-specific HOX-PBX transcriptional programs in pulmonary LAMCORE cells as regulators of cell survival depending upon HOXD11-PBX1 dimerization. Accordingly, blockage of HOXD11-PBX1 dimerization by HXR9 suppressed LAM cell survival in vitro and in vivo. PBX1 regulated STAT1/3, increased the expression of antiapoptotic genes, and promoted LAM cell survival in vitro. The HOX-PBX gene network provides promising targets for treatment of LAM/TSC mTORC1-hyperactive cancers.
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Redes Reguladoras de Genes , Proteínas de Homeodominio , Linfangioleiomiomatosis , Humanos , Análisis de la Célula Individual , Linfangioleiomiomatosis/metabolismo , Linfangioleiomiomatosis/patología , Factores de Transcripción/metabolismo , Pulmón/metabolismo , Pulmón/patología , Animales , Ratas , Metástasis de la Neoplasia , Multiómica , FemeninoRESUMEN
Pulmonary alveolar microlithiasis is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a pulmonary alveolar microlithiasis lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes and other proteins suggested a role for osteoclast-like cells in the host response to microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activation in a receptor activator of nuclear factor-κB ligand and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.