RESUMEN
Glioblastoma stem cells (GSCs) are implicated in tumor neovascularization, invasiveness, and therapeutic resistance. To illuminate mechanisms governing these hallmark features, we developed a de novo glioblastoma multiforme (GBM) model derived from immortalized human neural stem/progenitor cells (hNSCs) to enable precise system-level comparisons of pre-malignant and oncogene-induced malignant states of NSCs. Integrated transcriptomic and epigenomic analyses uncovered a PAX6/DLX5 transcriptional program driving WNT5A-mediated GSC differentiation into endothelial-like cells (GdECs). GdECs recruit existing endothelial cells to promote peritumoral satellite lesions, which serve as a niche supporting the growth of invasive glioma cells away from the primary tumor. Clinical data reveal higher WNT5A and GdECs expression in peritumoral and recurrent GBMs relative to matched intratumoral and primary GBMs, respectively, supporting WNT5A-mediated GSC differentiation and invasive growth in disease recurrence. Thus, the PAX6/DLX5-WNT5A axis governs the diffuse spread of glioma cells throughout the brain parenchyma, contributing to the lethality of GBM.
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Glioblastoma/genética , Glioblastoma/patología , Invasividad Neoplásica/genética , Proteína Wnt-5a/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Epigenómica , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Células-Madre Neurales/metabolismo , Factor de Transcripción PAX6/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Stem cells are fundamental units of tissue remodeling whose functions are dictated by lineage-specific transcription factors. Home to epidermal stem cells and their upward-stratifying progenies, skin relies on its secretory functions to form the outermost protective barrier, of which a transcriptional orchestrator has been elusive. KLF5 is a Krüppel-like transcription factor broadly involved in development and regeneration whose lineage specificity, if any, remains unclear. Here we report KLF5 specifically marks the epidermis, and its deletion leads to skin barrier dysfunction in vivo. Lipid envelopes and secretory lamellar bodies are defective in KLF5-deficient skin, accompanied by preferential loss of complex sphingolipids. KLF5 binds to and transcriptionally regulates genes encoding rate-limiting sphingolipid metabolism enzymes. Remarkably, skin barrier defects elicited by KLF5 ablation can be rescued by dietary interventions. Finally, we found that KLF5 is widely suppressed in human diseases with disrupted epidermal secretion, and its regulation of sphingolipid metabolism is conserved in human skin. Altogether, we established KLF5 as a disease-relevant transcription factor governing sphingolipid metabolism and barrier function in the skin, likely representing a long-sought secretory lineage-defining factor across tissue types.
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Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/patología , Ciclo Celular , Proteínas de Ciclo Celular , Línea Celular Tumoral , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Factores de Transcripción E2F/metabolismo , Humanos , Ratones , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP , Proteínas ras/metabolismoRESUMEN
Activating mutations in KRAS (KRAS*) are present in nearly all pancreatic ductal adenocarcinoma (PDAC) cases and critical for tumor maintenance. By using an inducible KRAS* PDAC mouse model, we identified a deubiquitinase USP21-driven resistance mechanism to anti-KRAS* therapy. USP21 promotes KRAS*-independent tumor growth via its regulation of MARK3-induced macropinocytosis, which serves to maintain intracellular amino acid levels for anabolic growth. The USP21-mediated KRAS* bypass, coupled with the frequent amplification of USP21 in human PDAC tumors, encourages the assessment of USP21 as a novel drug target as well as a potential parameter that may affect responsiveness to emergent anti-KRAS* therapy.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Enzimas Desubicuitinizantes/metabolismo , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ubiquitina TiolesterasaRESUMEN
In the mid-1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by a N-acetylglucosamine moiety (O-GlcNAc) via an O-linkage overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex, branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery, O-GlcNAcylation has been shown to contribute to numerous cellular functions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases, such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.
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Acetilglucosamina/genética , Fenómenos Fisiológicos Celulares/genética , N-Acetilglucosaminiltransferasas/genética , Procesamiento Proteico-Postraduccional/genética , Acetilglucosamina/metabolismo , Animales , Glicosilación , Humanos , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.
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Enfermedad de Alzheimer , Subunidades beta de la Proteína de Unión al GTP , Ratones , Humanos , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Estudio de Asociación del Genoma Completo , Ovillos Neurofibrilares/metabolismo , Fenotipo , Genómica , Péptidos beta-Amiloides/genética , Encéfalo/metabolismo , Miembro 5 de la Familia 22 de Transportadores de Solutos/genética , Miembro 5 de la Familia 22 de Transportadores de Solutos/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismoRESUMEN
To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-ß/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.
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Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Telomerasa/metabolismo , Telómero/metabolismo , Animales , Neoplasias Óseas/secundario , Línea Celular Tumoral , Cruzamientos Genéticos , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Femenino , Inestabilidad Genómica , Humanos , Masculino , Ratones , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Magnetic miniature robotic systems have attracted broad research interest because of their precise maneuverability in confined spaces and adaptability to diverse environments, holding significant promise for applications in both industrial infrastructures and biomedical fields. However, the predominant construction methodology involves the preprogramming of magnetic components into the system's structure. While this approach allows for intricate shape transformations, it exhibits limited flexibility in terms of reconfiguration and presents challenges when adapting to diverse materials, combining, and decoupling multiple functionalities. Here, we propose a construction strategy that facilitates the on-demand assembly of magnetic components, integrating ferrofluid droplets with the system's structural body. This approach enables the creation of complex solid-droplet robotic systems across a spectrum of length scales, ranging from 0.8 mm to 1.5 cm. It offers a diverse selection of materials and structural configurations, akin to assembling components like building blocks, thus allowing for the seamless integration of various functionalities. Moreover, it incorporates decoupling mechanisms to enable selective control over multiple functions, leveraging the fluidity, fission/fusion, and magneto-responsiveness properties inherent in the ferrofluid. Various solid-droplet systems have validated the feasibility of this strategy. This study advances the complexity and functionality achievable in small-scale magnetic robots, augmenting their potential for future biomedical and other applications.
RESUMEN
Immune checkpoint therapy has limited efficacy for patients with bone-metastatic castration-resistant prostate cancer (bmCRPC). To improve immunotherapy for bmCRPC, we aimed to identify the mechanism of bmCRPC-induced changes in the immune microenvironment. Among bmCRPC patients, higher levels of a 32-gene M2-like macrophage signature in bone metastasis samples correlated with shorter overall survival. Immunohistochemistry showed that CD206-positive (CD206+) macrophages were enriched in bmCRPC bone biopsy specimens compared with primary tumors or lymph node metastases. In preclinical osteogenic prostate cancer (Pca) xenograft models, CD206+ macrophages were recruited to areas with tumor-induced bone. RNA sequencing (RNAseq) analysis showed higher expression of an M2-like gene signature, with activated canonical and noncanonical Wnt pathways, in tumor-associated macrophages isolated from osteogenic tumors (bone-TAMs) than in TAMs isolated from nonosteogenic tumors (ctrl-TAMs). Mechanistic studies showed that endothelial cells (ECs) that had undergone EC-to-osteoblast (EC-to-OSB) transition, the precursors of tumor-induced OSBs, produced paracrine factors, including Wnts, CXCL14, and lysyl oxidase, which induced M2 polarization and recruited M2-like TAMs to the bone-tumor microenvironment (bone-TME). Bone-TAMs suppressed CD8+ T cells' proliferation and cytolytic activity, and these effects were partially reversed by treating bone-TAMs with Wnt inhibitors. Genetic or pharmacological inhibition of Pca-induced EC-to-OSB transition reduced the levels of M2-like macrophages in osteogenic tumors. Our study demonstrates that Pca-induced EC-to-OSB transition drives immunosuppression in the bone-TME, suggesting that therapies that reduce Pca-induced bone formation may improve immunotherapeutic outcomes for bmCRPC.
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Neoplasias Óseas , Células Endoteliales , Macrófagos , Osteoblastos , Microambiente Tumoral , Vía de Señalización Wnt , Masculino , Microambiente Tumoral/inmunología , Humanos , Neoplasias Óseas/inmunología , Neoplasias Óseas/secundario , Neoplasias Óseas/patología , Neoplasias Óseas/metabolismo , Animales , Ratones , Macrófagos/metabolismo , Macrófagos/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/inmunología , Osteoblastos/metabolismo , Osteoblastos/inmunología , Neoplasias de la Próstata Resistentes a la Castración/inmunología , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Línea Celular Tumoral , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/metabolismo , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
The ubiquitin-specific protease (USP) family is the largest group of cysteine proteases. Cancer genomic analysis identified frequent amplification of USP21 (22%) in human pancreatic ductal adenocarcinoma (PDAC). USP21 overexpression correlates with human PDAC progression, and enforced expression of USP21 accelerates murine PDAC tumor growth and drives PanIN to PDAC progression in immortalized human pancreatic ductal cells. Conversely, depletion of USP21 impairs PDAC tumor growth. Mechanistically, USP21 deubiquitinates and stabilizes the TCF/LEF transcription factor TCF7, which promotes cancer cell stemness. Our work identifies and validates USP21 as a PDAC oncogene, providing a potential druggable target for this intractable disease.
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Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Pancreáticas/enzimología , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Vía de Señalización Wnt/genética , Animales , Línea Celular Tumoral , Humanos , Ratones , Células Madre Neoplásicas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/fisiopatología , Factor 1 de Transcripción de Linfocitos T , Ubiquitinación , Neoplasias PancreáticasRESUMEN
Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Animales , Ratones , Fosfatidilinositol 3-Quinasas Clase III/genética , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Adipocitos/metabolismo , Lípidos , Tejido Adiposo Pardo/metabolismo , Adipocitos Marrones/metabolismoRESUMEN
In fungi, fusion between individuals leads to localized cell death, a phenomenon termed heterokaryon incompatibility. Generally, the genes responsible for this incompatibility are observed to be under balancing selection resulting from negative frequency-dependent selection. Here, we assess this phenomenon in Aspergillus fumigatus, a human pathogenic fungus with a very low level of linkage disequilibrium as well as an extremely high crossover rate. Using complementation of auxotrophic mutations as an assay for hyphal compatibility, we screened sexual progeny for compatibility to identify genes involved in this process, called het genes. In total, 5/148 (3.4%) offspring were compatible with a parent and 166/2,142 (7.7%) sibling pairs were compatible, consistent with several segregating incompatibility loci. Genetic mapping identified five loci, four of which could be fine mapped to individual genes, of which we tested three through heterologous expression, confirming their causal relationship. Consistent with long-term balancing selection, trans-species polymorphisms were apparent across several sister species, as well as equal allele frequencies within A. fumigatus. Surprisingly, a sliding window genome-wide population-level analysis of an independent dataset did not show increased Tajima's D near these loci, in contrast to what is often found surrounding loci under balancing selection. Using available de novo assemblies, we show that these balanced polymorphisms are restricted to several hundred base pairs flanking the coding sequence. In addition to identifying the first het genes in an Aspergillus species, this work highlights the interaction of long-term balancing selection with rapid linkage disequilibrium decay.
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Aspergillus fumigatus , Desequilibrio de Ligamiento , Selección Genética , Aspergillus fumigatus/genética , Genes Fúngicos , Frecuencia de los GenesRESUMEN
Low-temperature germination (LTG) is an important agronomic trait for direct-seeding cultivation of rice (Oryza sativa). Both OsMYB30 and OsTPP1 regulate the cold stress response in rice, but the function of OsMYB30 and OsTPP1 in regulating LTG and the underlying molecular mechanism remains unknown. Employing transcriptomics and functional studies revealed a sugar signaling pathway that regulates seed germination in response to low temperature (LT). Expression of OsMYB30 and OsTPP1 was induced by LT during seed germination, and overexpressing either OsMYB30 or OsTPP1 delayed seed germination and increased sensitivity to LT during seed germination. Transcriptomics and qPCR revealed that expression of OsTPP1 was upregulated in OsMYB30-overexpressing lines but downregulated in OsMYB30-knockout lines. In vitro and in vivo experiments revealed that OsMYB30 bound to the promoter of OsTPP1 and regulated the abundance of OsTPP1 transcripts. Overaccumulation of trehalose (Tre) was found in both OsMYB30- and OsTPP1-overexpressing lines, resulting in inhibition of α-amylase 1a (OsAMY1a) gene during seed germination. Both LT and exogenous Tre treatments suppressed the expression of OsAMY1a, and the osamy1a mutant was not sensitive to exogenous Tre during seed germination. Overall, we concluded that OsMYB30 expression was induced by LT to activate the expression of OsTPP1 and increase Tre content, which thus inhibited α-amylase activity and seed germination. This study identified a phytohormone-independent pathway that integrates environmental cues with internal factors to control seed germination.
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Oryza , Factores de Transcripción , Factores de Transcripción/genética , Germinación/genética , Trehalosa , alfa-Amilasas/genética , Temperatura , Semillas/genética , Oryza/genéticaRESUMEN
Desiccation is typically fatal, but a small number of land plants have evolved vegetative desiccation tolerance (VDT), allowing them to dry without dying through a process called anhydrobiosis. Advances in sequencing technologies have enabled the investigation of genomes for desiccation-tolerant plants over the past decade. However, a dedicated and integrated database for these valuable genomic resources has been lacking. Our prolonged interest in VDT plant genomes motivated us to create the "Drying without Dying" database, which contains a total of 16 VDT-related plant genomes (including 10 mosses) and incorporates 10 genomes that are closely related to VDT plants. The database features bioinformatic tools, such as blast and homologous cluster search, sequence retrieval, Gene Ontology term and metabolic pathway enrichment statistics, expression profiling, co-expression network extraction, and JBrowser exploration for each genome. To demonstrate its utility, we conducted tailored PFAM family statistical analyses, and we discovered that the drought-responsive ABA transporter AWPM-19 family is significantly tandemly duplicated in all bryophytes but rarely so in tracheophytes. Transcriptomic investigations also revealed that response patterns following desiccation diverged between bryophytes and angiosperms. Combined, the analyses provided genomic and transcriptomic evidence supporting a possible divergence and lineage-specific evolution of VDT in plants. The database can be accessed at http://desiccation.novogene.com. We expect this initial release of the "Drying without Dying" plant genome database will facilitate future discovery of VDT genetic resources.
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Briófitas , Desecación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Genoma de Planta/genética , Transcriptoma/genética , Briófitas/genéticaRESUMEN
Acute myeloid leukemia (AML) is an aggressive hematological malignancy. Nearly 50% of patients who receive the most intensive treatment inevitably experience disease relapse, likely resulting from the persistence of drug-resistant leukemia stem cells (LSCs). AML cells, especially LSCs, are highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for survival, but the mechanism involved in OXPHOS hyperactivity is unclear, and a noncytotoxic strategy to inhibit OXPHOS is lacking. To our knowledge, this study is the first to demonstrate that ZDHHC21 palmitoyltransferase serves as a key regulator of OXPHOS hyperactivity in AML cells. The depletion/inhibition of ZDHHC21 effectively induced myeloid differentiation and weakened stemness potential by inhibiting OXPHOS in AML cells. Interestingly, FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD)-mutated AML cells expressed significantly higher levels of ZDHHC21 and exhibited better sensitivity to ZDHHC21 inhibition. Mechanistically, ZDHHC21 specifically catalyzed the palmitoylation of mitochondrial adenylate kinase 2 (AK2) and further activated OXPHOS in leukemic blasts. Inhibition of ZDHHC21 arrested the in vivo growth of AML cells and extended the survival of mice inoculated with AML cell lines and patient derived xenograft AML blasts. Moreover, targeting ZDHHC21 to suppress OXPHOS markedly eradicated AML blasts and enhanced chemotherapy efficacy in relapsed/refractory leukemia. Together, these findings not only uncover a new biological function of palmitoyltransferase ZDHHC21 in regulating AML OXPHOS but also indicate that ZDHHC21 inhibition is a promising therapeutic regimen for patients with AML, especially relapsed/refractory leukemia.
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Leucemia Mieloide Aguda , Fosforilación Oxidativa , Animales , Humanos , Ratones , Diferenciación Celular , Tirosina Quinasa 3 Similar a fms/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutación , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.
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Células Secretoras de Insulina , Insulina , Vesículas Secretoras , Zinc , Animales , Glucemia , Línea Celular , Insulina/análisis , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestructura , Ratas , Vesículas Secretoras/química , Vesículas Secretoras/metabolismo , Espectrometría por Rayos X , Difracción de Rayos X , Zinc/análisisRESUMEN
Human colorectal cancer (CRC) is a major cause of cancer mortality and frequently harbors activating mutations in the KRAS gene. To understand the role of oncogenic KRAS in CRC, we engineered a mouse model of metastatic CRC that harbors an inducible oncogenic Kras allele (Krasmut ) and conditional null alleles of Apc and Trp53 (iKAP). The iKAP model recapitulates tumor progression from adenoma through metastases. Whole-exome sequencing revealed that the Krasmut allele was heterogenous in primary tumors yet homogenous in metastases, a pattern consistent with activated Krasmut signaling being a driver of progression to metastasis. System-level and functional analyses revealed the TGF-ß pathway as a key mediator of Krasmut -driven invasiveness. Genetic extinction of Krasmut resulted in specific elimination of the Krasmut subpopulation in primary and metastatic tumors, leading to apoptotic elimination of advanced invasive and metastatic disease. This faithful CRC model provides genetic evidence that Krasmut drives CRC invasion and maintenance of metastases.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/fisiopatología , Invasividad Neoplásica/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Genotipo , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas p21(ras)/genética , Transcriptoma , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Remuscularization of the mammalian heart can be achieved after cell transplantation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). However, several hurdles remain before implementation into clinical practice. Poor survival of the implanted cells is related to insufficient vascularization, and the potential for fatal arrhythmogenesis is associated with the fetal cell-like nature of immature CMs. METHODS: We generated 3 lines of hiPSC-derived endothelial cells (ECs) and hiPSC-CMs from 3 independent donors and tested hiPSC-CM sarcomeric length, gap junction protein, and calcium-handling ability in coculture with ECs. Next, we examined the therapeutic effect of the cotransplantation of hiPSC-ECs and hiPSC-CMs in nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice undergoing myocardial infarction (n≥4). Cardiac function was assessed by echocardiography, whereas arrhythmic events were recorded using 3-lead ECGs. We further used healthy non-human primates (n=4) with cell injection to study the cell engraftment, maturation, and integration of transplanted hiPSC-CMs, alone or along with hiPSC-ECs, by histological analysis. Last, we tested the cell therapy in ischemic reperfusion injury in non-human primates (n=4, 3, and 4 for EC+CM, CM, and control, respectively). Cardiac function was evaluated by echocardiography and cardiac MRI, whereas arrhythmic events were monitored by telemetric ECG recorders. Cell engraftment, angiogenesis, and host-graft integration of human grafts were also investigated. RESULTS: We demonstrated that human iPSC-ECs promote the maturity and function of hiPSC-CMs in vitro and in vivo. When cocultured with ECs, CMs showed more mature phenotypes in cellular structure and function. In the mouse model, cotransplantation augmented the EC-accompanied vascularization in the grafts, promoted the maturity of CMs at the infarct area, and improved cardiac function after myocardial infarction. Furthermore, in non-human primates, transplantation of ECs and CMs significantly enhanced graft size and vasculature and improved cardiac function after ischemic reperfusion. CONCLUSIONS: These results demonstrate the synergistic effect of combining iPSC-derived ECs and CMs for therapy in the postmyocardial infarction heart, enabling a promising strategy toward clinical translation.
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Células Madre Pluripotentes Inducidas , Infarto del Miocardio , Humanos , Ratones , Animales , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Endoteliales/metabolismo , Ratones SCID , Ratones Endogámicos NOD , Infarto del Miocardio/patología , Primates , Diferenciación Celular , MamíferosRESUMEN
Communication between cells is crucial to the survival of both uni- and multicellular organisms. The primary mode of communication involves chemical cues. There is great current interest in mimicking this behavior in synthetic cells to understand the physical basis of intercellular communication and design collective functional behavior. Using liposomal cell mimics, we demonstrate how a chemical input can elicit a mechanical response (enhanced motility). We employed a single substrate to trigger enzyme cascade-induced control of the diffusion of up to three different liposome populations. Furthermore, substrate competition allows temporal control over enhanced diffusion. The use of enzyme cascades to propagate chemical signals provides a robust and efficient mechanism for diverse populations of protocells to coordinate their motion in response to signals from each other.