RESUMO
Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and adolescents. Fusion-negative RMS (FN-RMS) accounts for more than 80% of all RMS cases. The long-term event-free survival rate for patients with high-grade FN-RMS is below 30%, highlighting the need for improved therapeutic strategies. CD73 is a 5' ectonucleotidase that hydrolyzes AMP to adenosine and regulates the purinergic signaling pathway. We found that CD73 is elevated in FN-RMS tumors that express high levels of TWIST2. While high expression of CD73 contributes to the pathogenesis of multiple cancers, its role in FN-RMS has not been investigated. We found that CD73 knockdown decreased FN-RMS cell growth while up-regulating the myogenic differentiation program. Moreover, mutation of the catalytic residues of CD73 rendered the protein enzymatically inactive and abolished its ability to stimulate FN-RMS growth. Overexpression of wildtype CD73, but not the catalytically inactive mutant, in CD73 knockdown FN-RMS cells restored their growth capacity. Likewise, treatment with an adenosine receptor A2A-B agonist partially rescued FN-RMS cell proliferation and bypassed the CD73 knockdown defective growth phenotype. These results demonstrate that the catalytic activity of CD73 contributes to the pathogenic growth of FN-RMS through the activation of the purinergic signaling pathway. Therefore, targeting CD73 and the purinergic signaling pathway represents a potential therapeutic approach for FN-RMS patients.
Assuntos
Rabdomiossarcoma , Adolescente , Criança , Humanos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Receptores Purinérgicos P1 , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Transdução de SinaisRESUMO
Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.
Assuntos
Carcinogênese , Desenvolvimento Muscular , Proteína MyoD/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Células Cultivadas , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Transição Epitelial-Mesenquimal , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Sequências Hélice-Alça-Hélice , Humanos , Proteína MyoD/química , Mioblastos/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/química , Proteína 1 Relacionada a Twist/químicaRESUMO
Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early T cell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish T cell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid-tissue-inducer-like cells. E2A and HEB orchestrated T cell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, T cell receptor (TCR) assembly, and TCR-mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T-cell-lineage-specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E-Id protein axis specifies innate and adaptive lymphoid cell fate.
Assuntos
Imunidade Adaptativa , Imunidade Inata , Imunomodulação , Subpopulações de Linfócitos/imunologia , Timócitos/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/imunologia , Análise por Conglomerados , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Imunofenotipagem , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/metabolismo , Células Progenitoras Linfoides/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Timócitos/citologia , Timócitos/metabolismo , TranscriptomaRESUMO
BACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic ß-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. CONCLUSIONS: Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.
Assuntos
Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Camundongos , Humanos , Animais , Edição de Genes , Sistemas CRISPR-Cas , Camundongos Knockout , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação , Fibrose , Adenina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismoRESUMO
BACKGROUND & AIMS: Thirty to 90% of hepatocytes contain whole-genome duplications, but little is known about the fates or functions of these polyploid cells or how they affect development of liver disease. We investigated the effects of continuous proliferative pressure, observed in chronically damaged liver tissues, on polyploid cells. METHODS: We studied Rosa-rtTa mice (controls) and Rosa-rtTa;TRE-short hairpin RNA mice, which have reversible knockdown of anillin, actin binding protein (ANLN). Transient administration of doxycycline increases the frequency and degree of hepatocyte polyploidy without permanently altering levels of ANLN. Mice were then given diethylnitrosamine and carbon tetrachloride (CCl4) to induce mutations, chronic liver damage, and carcinogenesis. We performed partial hepatectomies to test liver regeneration and then RNA-sequencing to identify changes in gene expression. Lineage tracing was used to rule out repopulation from non-hepatocyte sources. We imaged dividing hepatocytes to estimate the frequency of mitotic errors during regeneration. We also performed whole-exome sequencing of 54 liver nodules from patients with cirrhosis to quantify aneuploidy, a possible outcome of polyploid cell divisions. RESULTS: Liver tissues from control mice given CCl4 had significant increases in ploidy compared with livers from uninjured mice. Mice with knockdown of ANLN had hepatocyte ploidy above physiologic levels and developed significantly fewer liver tumors after administration of diethylnitrosamine and CCl4 compared with control mice. Increased hepatocyte polyploidy was not associated with altered regenerative capacity or tissue fitness, changes in gene expression, or more mitotic errors. Based on lineage-tracing experiments, non-hepatocytes did not contribute to liver regeneration in mice with increased polyploidy. Despite an equivalent rate of mitosis in hepatocytes of differing ploidies, we found no lagging chromosomes or micronuclei in mitotic polyploid cells. In nodules of human cirrhotic liver tissue, there was no evidence of chromosome-level copy number variations. CONCLUSIONS: Mice with increased polyploid hepatocytes develop fewer liver tumors following chronic liver damage. Remarkably, polyploid hepatocytes maintain the ability to regenerate liver tissues during chronic damage without generating mitotic errors, and aneuploidy is not commonly observed in cirrhotic livers. Strategies to increase numbers of polypoid hepatocytes might be effective in preventing liver cancer.
Assuntos
Carcinoma Hepatocelular/genética , Hepatócitos/fisiologia , Neoplasias Hepáticas/genética , Regeneração Hepática/genética , Poliploidia , Animais , Tetracloreto de Carbono/toxicidade , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Dietilnitrosamina/toxicidade , Feminino , Técnicas de Silenciamento de Genes , Hepatectomia , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Cultura Primária de Células , Fatores de Proteção , RNA-Seq , Sequenciamento do ExomaRESUMO
Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.
Assuntos
Proteínas de Homeodomínio/metabolismo , Tecido Parenquimatoso/fisiologia , Dente/citologia , Dente/embriologia , Adolescente , Animais , Feminino , Proteínas de Homeodomínio/genética , Humanos , Incisivo/citologia , Incisivo/embriologia , Camundongos Endogâmicos , Dente Serotino/citologia , Técnicas de Cultura de Órgãos , Tecido Parenquimatoso/citologia , Gravidez , Regiões Promotoras Genéticas , Regeneração , Células Estromais/fisiologia , Suínos , Fator A de Crescimento do Endotélio Vascular/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismoRESUMO
Multiple myeloma (MM) is an aggressive cancer that originates from antibody-secreting plasma cells. Although genetically and transcriptionally well characterized, the aberrant gene regulatory networks that underpin this disease remain poorly understood. Here, we mapped regulatory elements, open chromatin, and transcription factor (TF) footprints in primary MM cells. In comparison with normal antibody-secreting cells, MM cells displayed consistent changes in enhancer activity that are connected to superenhancer (SE)-mediated deregulation of TF genes. MM cells also displayed widespread decompaction of heterochromatin that was associated with activation of regulatory elements and in a major subset of patients' deregulation of the cyclic adenosine monophosphate pathway. Finally, building SE-associated TF-based regulatory networks allowed identification of several novel TFs that are central to MM biology. Taken together, these findings significantly add to our understanding of the aberrant gene regulatory network that underpins MM.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Mieloma Múltiplo/genética , Biomarcadores , Linhagem da Célula/genética , Cromatina/metabolismo , Biologia Computacional/métodos , Humanos , Imunofenotipagem , Mieloma Múltiplo/metabolismo , Translocação GenéticaRESUMO
Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue- or cell type-specific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23-42% of all cassette exons under tissue- or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome.
Assuntos
Processamento Alternativo/genética , Encéfalo/metabolismo , Cromatina/metabolismo , Sequência Conservada/genética , Éxons/genética , Mamíferos/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Animais , Sequência de Bases , Córtex Cerebral/metabolismo , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Especificidade de Órgãos/genética , Seleção Genética , Transcriptoma/genéticaRESUMO
Long non-coding RNAs (lncRNAs) (> 200 bp) play crucial roles in transcriptional regulation during numerous biological processes. However, it is challenging to comprehensively identify lncRNAs, because they are often expressed at low levels and with more cell-type specificity than are protein-coding genes. In the present study, we performed ab initio transcriptome reconstruction using eight purified cell populations from mouse cortex and detected more than 5000 lncRNAs. Predicting the functions of lncRNAs using cell-type specific data revealed their potential functional roles in Central Nervous System (CNS) development. We performed motif searches in ENCODE DNase I digital footprint data and Mouse ENCODE promoters to infer transcription factor (TF) occupancy. By integrating TF binding and cell-type specific transcriptomic data, we constructed a novel framework that is useful for systematically identifying lncRNAs that are potentially essential for brain cell fate determination. Based on this integrative analysis, we identified lncRNAs that are regulated during Oligodendrocyte Precursor Cell (OPC) differentiation from Neural Stem Cells (NSCs) and that are likely to be involved in oligodendrogenesis. The top candidate, lnc-OPC, shows highly specific expression in OPCs and remarkable sequence conservation among placental mammals. Interestingly, lnc-OPC is significantly up-regulated in glial progenitors from experimental autoimmune encephalomyelitis (EAE) mouse models compared to wild-type mice. OLIG2-binding sites in the upstream regulatory region of lnc-OPC were identified by ChIP (chromatin immunoprecipitation)-Sequencing and validated by luciferase assays. Loss-of-function experiments confirmed that lnc-OPC plays a functional role in OPC genesis. Overall, our results substantiated the role of lncRNA in OPC fate determination and provided an unprecedented data source for future functional investigations in CNS cell types. We present our datasets and analysis results via the interactive genome browser at our laboratory website that is freely accessible to the research community. This is the first lncRNA expression database of collective populations of glia, vascular cells, and neurons. We anticipate that these studies will advance the knowledge of this major class of non-coding genes and their potential roles in neurological development and diseases.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Córtex Cerebral/crescimento & desenvolvimento , RNA Longo não Codificante/genética , Transcriptoma/genética , Animais , Córtex Cerebral/metabolismo , Sequência Conservada/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma , Camundongos , Neurônios/metabolismo , Oligodendroglia/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/classificaçãoRESUMO
The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.
Assuntos
Processamento Alternativo , Córtex Cerebral/metabolismo , Bases de Dados de Ácidos Nucleicos , Endotélio Vascular/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Transcriptoma , Animais , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/citologia , Camundongos , Análise de Sequência de RNARESUMO
Tendon injuries often lead to joint dysfunction due to the limited self-regeneration capacity of tendons. Repairing tendons is a major challenge for surgeons and imposes a significant financial burden on society. Therefore, there is an urgent need to develop effective strategies for repairing injured tendons. Tendon tissue engineering using hydrogels has emerged as a promising approach that has attracted considerable interest. Hydrogels possess excellent biocompatibility and biodegradability, enabling them to create an extracellular matrix-like growth environment for cells. They can also serve as a carrier for cells or other substances to accelerate tendon repair. In the past decade, numerous studies have made significant progress in the preparation of hydrogel scaffolds for tendon healing. This review aims to provide an overview of recent research on the materials of hydrogel-based scaffolds used for tendon tissue engineering and discusses the delivery systems based on them.
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Wilms tumor is the most common kidney cancer in children, and diffusely anaplastic Wilms tumor is the most chemoresistant histological subtype. Here we explore how Wilms tumor cells evade the common chemotherapeutic drug actinomycin D, which inhibits ribosomal RNA biogenesis. Using ribosome profiling, protein arrays, and a genome-wide knockout screen, we describe how actinomycin D disrupts protein homeostasis and blocks cell cycle progression. We found that, when ribosomal capacity is limited by actinomycin D treatment, anaplastic Wilms tumor cells preferentially translate proteasome components and upregulate proteasome activity. Furthermore, the proteasome inhibitor bortezomib sensitizes cells to actinomycin D treatment by inducing apoptosis both in vitro and in vivo. Lastly, we show that increased levels of proteasome components are associated with anaplastic histology and with worse prognosis in non-anaplastic Wilms tumor. In sum, maintaining protein homeostasis is critical for Wilms tumor proliferation, and it can be therapeutically disrupted by blocking protein synthesis or turnover.
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BACKGROUND: Treatment options for calcific tendinitis (CT) of the shoulder remain controversial. A consensus for an operative indication for this condition is lacking. PURPOSE: To compare nonoperative versus operative treatment for shoulder CT and analyze factors affecting the prognosis after treatment. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A total of 180 patients diagnosed with symptomatic CT between January 2017 and September 2021 were evaluated in this retrospective cohort study. There were 103 patients treated nonoperatively at our institution, which included the use of nonsteroidal anti-inflammatory drugs, acupuncture, steroid injections, extracorporeal shock wave therapy, and ultrasound-guided needle aspiration/percutaneous irrigation. However, 77 patients were treated with arthroscopic surgery after 6 months of failed nonoperative treatment. The visual analog scale (VAS) for pain, the Constant-Murley score, and imaging were used to assess and evaluate outcomes. Descriptive data, functional outcomes, and imaging findings were compared between the operative and nonoperative groups before and after propensity score matching. Additionally, prognostic factors including calcium deposit size, tendon infiltration by calcium deposits, involvement of single or multiple tendons, and occurrence of rotator cuff tears were analyzed by comparing the groups to determine their effect on treatment options and recovery. RESULTS: Magnetic resonance imaging showed that the supraspinatus tendon (66.7%) was most commonly involved, followed by the infraspinatus (42.8%) and subscapularis (21.1%) tendons. Tendon infiltration by calcium deposits was observed in 84.4% of the patients, and rotator cuff tears occurred in 30.0% of the patients. After propensity score matching, there was no significant difference in changes in the Constant-Murley score (48.1 ± 25.4 vs 49.0 ± 22.8, respectively; P = .950) and VAS score (4.9 ± 2.3 vs 4.5 ± 1.9, respectively; P = .860) between the operative and nonoperative groups at the final follow-up. However, for patients with shoulder CT and without rotator cuff tears, there was a significant difference in changes in the Constant-Murley score (52.93 ± 25.18 vs 42.13 ± 22.35, respectively; P = .012) and VAS score (5.21 ± 2.06 vs 3.81 ± 1.98, respectively; P < .001) between the operative and nonoperative groups, but the recovery time in the operative group was longer than that in the nonoperative group (86.92 ± 138.56 vs 30.42 ± 54.97 days, respectively; P = .016). The results also showed that calcium deposit size, involvement of multiple tendons, and tendon infiltration by calcium deposits did not affect the recovery time after treatment. The survival analysis showed that rotator cuff tears affected the complete recovery of shoulder function. CONCLUSION: This study demonstrated no significant difference between nonoperative and operative treatment for patients with shoulder CT, on the whole. However, for patients with shoulder CT and without rotator cuff tears, the effect of operative treatment was better than that of nonoperative treatment; yet, operative treatment was shown to prolong the recovery time. Calcium deposit size, tendon infiltration by calcium deposits, and involvement of multiple tendons did not correlate with recovery time or the recovery of function. A rotator cuff tear was the only factor affecting the complete recovery of shoulder function.
Assuntos
Lesões do Manguito Rotador , Tendinopatia , Humanos , Ombro/cirurgia , Lesões do Manguito Rotador/diagnóstico por imagem , Lesões do Manguito Rotador/cirurgia , Estudos de Coortes , Artroscopia/métodos , Estudos Retrospectivos , Cálcio , Resultado do Tratamento , Imageamento por Ressonância Magnética , Tendinopatia/diagnóstico por imagem , Tendinopatia/terapiaRESUMO
Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.
Assuntos
Cardiomiopatias , Doenças Cardiovasculares , Camundongos , Animais , Humanos , Sistemas CRISPR-Cas , Edição de Genes , Coração , Cardiomiopatias/genética , Doenças Cardiovasculares/genéticaRESUMO
Cancer cachexia is a prevalent and often fatal wasting condition that cannot be fully reversed with nutritional interventions. Muscle atrophy is a central component of the syndrome, but the mechanisms whereby cancer leads to skeletal muscle atrophy are not well understood. We performed single-nucleus multi-omics on skeletal muscles from a mouse model of cancer cachexia and profiled the molecular changes in cachexic muscle. Our results revealed the activation of a denervation-dependent gene program that upregulates the transcription factor myogenin. Further studies showed that a myogenin-myostatin pathway promotes muscle atrophy in response to cancer cachexia. Short hairpin RNA inhibition of myogenin or inhibition of myostatin through overexpression of its endogenous inhibitor follistatin prevented cancer cachexia-induced muscle atrophy in mice. Our findings uncover a molecular basis of muscle atrophy associated with cancer cachexia and highlight potential therapeutic targets for this disorder.
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Caquexia , Atrofia Muscular , Miogenina , Miostatina , Caquexia/patologia , Caquexia/metabolismo , Caquexia/etiologia , Animais , Atrofia Muscular/patologia , Atrofia Muscular/metabolismo , Camundongos , Miostatina/metabolismo , Miostatina/genética , Miogenina/metabolismo , Miogenina/genética , Músculo Esquelético/patologia , Músculo Esquelético/metabolismo , Neoplasias/complicações , Neoplasias/patologia , Neoplasias/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Transdução de Sinais , Folistatina/metabolismo , HumanosRESUMO
During development, cells make switch-like decisions to activate new gene programs specifying cell lineage. The mechanisms underlying these decisive choices remain unclear. Here, we show that the cardiovascular transcriptional coactivator myocardin (MYOCD) activates cell identity genes by concentration-dependent and switch-like formation of transcriptional condensates. MYOCD forms such condensates and activates cell identity genes at critical concentration thresholds achieved during smooth muscle cell and cardiomyocyte differentiation. The carboxyl-terminal disordered region of MYOCD is necessary and sufficient for condensate formation. Disrupting this region's ability to form condensates disrupts gene activation and smooth muscle cell reprogramming. Rescuing condensate formation by replacing this region with disordered regions from functionally unrelated proteins rescues gene activation and smooth muscle cell reprogramming. Our findings demonstrate that MYOCD condensate formation is required for gene activation during cardiovascular differentiation. We propose that the formation of transcriptional condensates at critical concentrations of cell type-specific regulators provides a molecular switch underlying the activation of key cell identity genes during development.
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Miócitos de Músculo Liso , Fatores de Transcrição , Linhagem da Célula/genética , Diferenciação Celular/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Miócitos de Músculo Liso/metabolismo , Ativação TranscricionalRESUMO
We present an integrated single-cell RNA sequencing atlas of the primary breast tumor microenvironment (TME) containing 236,363 cells from 119 biopsy samples across eight datasets. In this study, we leverage this resource for multiple analyses of immune and cancer epithelial cell heterogeneity. We define natural killer (NK) cell heterogeneity through six subsets in the breast TME. Because NK cell heterogeneity correlates with epithelial cell heterogeneity, we characterize epithelial cells at the level of single-gene expression, molecular subtype, and 10 categories reflecting intratumoral transcriptional heterogeneity. We develop InteractPrint, which considers how cancer epithelial cell heterogeneity influences cancer-immune interactions. We use T cell InteractPrint to predict response to immune checkpoint inhibition (ICI) in two breast cancer clinical trials testing neoadjuvant anti-PD-1 therapy. T cell InteractPrint was predictive of response in both trials versus PD-L1 (AUC = 0.82, 0.83 vs. 0.50, 0.72). This resource enables additional high-resolution investigations of the breast TME.
Assuntos
Neoplasias da Mama , Inibidores de Checkpoint Imunológico , Células Matadoras Naturais , Análise de Célula Única , Microambiente Tumoral , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Feminino , Microambiente Tumoral/imunologia , Análise de Célula Única/métodos , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Células Matadoras Naturais/imunologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Regulação Neoplásica da Expressão Gênica , Linfócitos T/imunologia , Heterogeneidade GenéticaRESUMO
There are few effective treatments for small cell lung cancer (SCLC) underscoring the need for innovative therapeutic approaches. This study focuses on exploiting telomerase, a critical SCLC dependency as a therapeutic target. A prominent characteristic of SCLC is their reliance on telomerase activity, a key enzyme essential for their continuous proliferation. Here we utilize a nucleoside analog, 6-Thio-2'-deoxyguanosine (6TdG) currently in phase II clinical trials, that is preferentially incorporated by telomerase into telomeres leading to telomere dysfunction. Using preclinical mouse and human derived models we find low intermittent doses of 6TdG inhibit tumor growth and reduce metastatic burden. Anti-tumor efficacy correlates with a reduction in a subpopulation of cancer initiating like cells (CICs) identified by their expression of L1CAM/CD133 and highest telomerase activity. 6TdG treatment also leads to activation of innate and adaptive anti-tumor responses. Mechanistically, 6TdG depletes CICs and induces type-I interferon signaling leading to tumor immune visibility by activating tumor cell STING signaling. We also observe increased sensitivity to irradiation after 6TdG treatment in both syngeneic and humanized SCLC xenograft models both of which are dependent on the presence of host immune cells. This study underscores the immune-enhancing and metastasis-reducing effects of 6TdG, employing a range of complementary in vitro and in vivo SCLC preclinical models providing a potential therapeutic approach to SCLC.
Assuntos
Desoxiguanosina/análogos & derivados , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Telomerase , Tionucleosídeos , Humanos , Animais , Camundongos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Sistemas de Liberação de Medicamentos , TelômeroRESUMO
Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that causes significant devastation, with no effective therapy for relapsed disease. The mechanisms behind treatment failures are poorly understood. Our study showed that treatment of RMS cells with vincristine led to an increase in CD133-positive stem-like resistant cells. Single cell RNAseq analysis revealed that MYC and YBX1 were among the top-scoring transcription factors in CD133-high expressing cells. Targeting MYC and YBX1 using CRISPR/Cas9 reduced stem-like characteristics and viability of the vincristine-resistant cells. MYC and YBX1 showed mutual regulation, with MYC binding to the YBX1 promoter and YBX1 binding to MYC mRNA. The MYC inhibitor MYC361i synergized with vincristine to reduce tumor growth and stem-like cells in a zebrafish model of RMS. MYC and YBX expression showed a positive correlation in RMS patients, and high MYC expression correlated with poor survival. Targeting the MYC-YBX1 axis holds promise for improving survival in RMS patients.
RESUMO
CRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIIδ, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIIδ gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIIδ editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIIδ gene editing may thus represent a permanent and advanced strategy for heart disease therapy.